The evolution of nervous systems has included significant changes in the axon tracts of the central nervous system. These evolutionary changes required changes in axonal growth in embryos. During development, many axons reach their targets by following guidance cues that are organized as pathways in the embryonic substrate, and the overall pattern of the major axon tracts in the adult can be traced back to the fundamental pattern of such substrate pathways. Embryological and comparative anatomical studies suggest that most axon tracts, such as the anterior commissure, have evolved by the modified use of preexisting substrate pathways. On the other hand, recent developmental studies suggest that a few entirely new substrate pathways have arisen during evolution; these apparently provided opportunities for the formation of completely new axon tracts. The corpus callosum, which is found only in placental mammals, may be such a truly new axon tract. We propose that the evolution of the corpus callosum is founded on the emergence of a new preaxonal substrate pathway, the "glial sling," which bridges the two halves of the embryonic forebrain only in placental mammals.Increases in the number of central nervous system (CNS) neurons and in the complexity of the synaptic neuropil characterize the vertebrate phylogeny (1, 2). These increases are usually manifest as enlargements of specific CNS areas such as the cerebellum, the tectum, and the cerebrum. Local enlargements can be attributed to focal increases in the dominant class of vertebrate neurons-local circuit neurons-neurons with axons that extend only short distances in the CNS before synapsing (1).Local areas of the CNS are connected by another important class of neurons-projection neurons-which send their axons along a few long stereotyped routes to target areas far from their cell bodies. The white matter of the vertebrate CNS is composed largely of axon tracts of projection neurons, and many of the same major axon tracts can be identified throughout the vertebrates. Although the overall pattern of these major tracts appears to have been conserved during evolution (2-8), there are some significant variations. Frequently, the compactness of particular tracts varies, as in the spinal lemniscal tracts (2, 9-11). In some cases, the particular stereotyped route taken by homologous axons varies-e.g., the stereotyped routes of the corticospinal tracts differ among most primates, ungulates, rodents, and marsupials (12-14). Moreover, a few major vertebrate axon tracts have appeared with no apparent precursors. The most dramatic example is the (dorsal) corpus callosum, which is found only in placental mammals (15,16).The corpus callosum is a large interhemispheric commissure, and most axons of the corpus callosum interconnect homotopic neocortical areas (17)(18)(19)(20)(21)(22). for exceptions to this general rule.) Monotremes and marsupials have no dorsal corpus callosum (2,16,(26)(27)(28). Among the placental mammals, the corpus callosum generally increases as t...