(Top) The pathfinding behavior of commissural axons was visualized through the application of DiI into a dorsal region of fixed, open-book spinal cord preparations taken from E12.5 embryos. (A-F) Photomicrographs of DiI-labeled axons in open book preparations derived from wild type (WT) and Math1/L1 embryos. (A, D) In WT embryos, commissural axons project directly through the FP in a well-organized fashion before turning in a predominantly rostral direction on the contralateral side of the spinal cord. (B, C, E) In Math1/L1 embryos, commissural axons “pile-up” and stall at the ipsilateral margin of the FP, or project along this boundary before ultimately crossing the FP. The arrowheads in A, B and C denote out of focus axons that are projecting along appropriately shaped contralateral trajectories in WT and Math1/L1 embryos. (F) On rare occasions, some commissural axons were observed to stall at the contralateral margin of the FP. In A-F, asterisks demarcate the position of the ipsilateral FP margin. In each panel, rostral is to the right. Scale bar in (F), 95 μm (A-C); 50 μm (D-F).