Slit2 guides both precrossing and postcrossing callosal axons at the midline in vivo.

Commissural axons generally cross the midline only once. In the Drosophila nerve cord and mouse spinal cord, commissural axons are guided by Slit only after they cross the midline, where Slit prevents these axons from recrossing the midline. In the developing corpus callosum, Slit2 expressed by the glial wedge guides callosal axons before they cross the midline, as they approach the corticoseptal boundary. These data highlighted a potential difference between the role of Slit2 in guiding commissural axons in the brain compared with the spinal cord. Here, we investigate whether Slit2 also guides callosal axons after they cross the midline. Because such questions cannot be addressed in conventional gene knock-out animals, we used in utero injections of antisense oligonucleotides to specifically deplete Slit2 on only one side of the brain. We used this technique together with a novel in vitro assay of hemisected brain slices to specifically analyze postcrossing callosal axons. We find that in the brain, unlike the spinal cord, Slit2 mediates both precrossing and postcrossing axonal guidance. Depletion of Slit2 on one side of the brain causes axons to defasciculate and, in some cases, to aberrantly enter the septum. Because these axons do not recross the midline, we conclude that the principle function of Slit2 at the cortical midline may be to channel the axons along the correct path and possibly repel them away from the midline. We find no evidence that Slit2 prevents axons from recrossing the midline in the brain.