Fig. 3. The glial wedge directs the growth of callosal axons in situ. A, The glial wedge forms in organotypic slices. E17 mouse brains were sectioned at 350 μm, grown for 3 d in culture, fixed, resectioned at 50 μm, and stained with a GFAP antibody. Both the glial wedge (arrow) and the indusium griseum glia (arrowhead) maintain their in vivomorphology after 3 d in vitro. B,C, The corpus callosum forms in organotypic slices.B and C represent two examples of uncut control slices, cultured for 3 d and fixed, and a crystal of DiI was added to label the callosal axons. In C, DiI crystals were placed on both sides of the midline to show that cortical axons from both hemispheres still cross in the same organotypic slice (in all other slices, DiI was added to only one hemisphere).D–L, Replacement or reorientation of the midline results in axonal misrouting. D–F, The corpus callosum forms normally in sham-operated slices (D shows the experimental paradigm, and E and F are two examples). G–I, Reorienting the glial wedge by 180° causes the axons to turn away from the midline (Gshows the experimental paradigm, and H andI are two examples). J–L, The glial wedge is required for axons to turn toward the midline. When the glial wedge–indusium griseum region is replaced on one side by a piece of cortex, cortical axons fail to turn and instead grow straight through the graft, in many cases entering the septum (arrow inK; J shows the experimental paradigm, andK and L are two examples). Thewhite broken lines in B,C, E, F, H, and I represent the position of the glial wedge; inK and L, they represent the edges of the cortical graft. The solid white line inB, C, E, F,H, I, K, andL represent the position of the midline. Scale bar (inL): A, 120 μm; C,E, F, K, L, 200 μm; B, H, I, 100 μm.