DCNsrely on Robo/Slit signaling at the dorsal midline but utilize aRobo3-independent midline crossing mechanism. (A–B′)
Slit1 and Slit2 are expressed at the dorsal (red arrowheads) and ventral (orange arrowheads) midline in E15.5 spinal cord. The dorsal midline is magnified in (A′) and (B′). (C–H) At E15.5, Robo1 and Robo2 are expressed in dorsolateral spinal cord (C,D) and DRG (F,G). Robo3 is sparsely expressed in the dorsolateral spinal cord (E) and is absent from DRG (H). (I–L) L1CAMON ectopic longitudinal funiculi [arrowheads in (L)] are present proximally to the dorsal midline in the Robo1/2 double mutant at E16.5. (J) corresponds to the boxed region in (I). (L) corresponds to boxed region in (K). (M–N′) In longitudinal sections, L1CAMON misguided axons are present at the dorsoventral level of the dorsal commissure and diverge as they approach the dorsal midline, likely comprising the ectopic longitudinal funiculi present in transverse sections. (M′) and (N′) represent tracings of control and misguided axons, respectively. (O,O′) L1CAMON DCN axons persist in E16.5 Robo3 mutant spinal cords. (O′) corresponds to boxed region in (O). (P,Q) DiI-labeled DCN axons (blue arrowhead) cross the midline in E16.5 Robo3 mutants, unlike ventrally-crossing commissural axons (orange arrowhead), which fail to cross the midline (Q; Sabatier et al., 2004). (R) Summary of findings. VCN, ventrally-crossing commissural neurons. Scale bars: 100 μm (A,B,C–E,I,K,M–O,P,Q); 50 μm (A′,B′,O′,F–H); 25 μm (J,L).