A large number of zebrafish retinotectal mutants, in which axons from the eye fail to reach their proper targets in the brain, are important tools for investigating the mechanisms that guide axons across in the developing brain. We showed that the “achiasmatic” mutant belladonna (bel) affects the lhx2 gene and that in both lhx2 and shh mutants the axon growth substrate is disrupted in specific ways. This analysis is providing clues as to the guidance mechanisms that are required for proper midline crossing. In particular, we have characterized a unique population of glial cells that spans the midline just prior to axon crossing, and shown that this glial bridge is disrupted in the achiasmatic mutants. We are now interested in understanding how these glial cells find their correct positions in the forebrain and how they subsequently help establish of the optic chiasm and forebrain commissures.