Stereotyped pathway selection by growth cones of early epiphysial neurons in the embryonic zebrafish

In this report we have examined the development of one of the earliest projections in the embryonic zebrafish brain, that from the epiphysis. Epiphysial axons and growth cones were labelled anterogradely in whole-mounted brains, using either the carbocyanine dye, diI, or horseradish peroxidase (HRP). Some embryos were also either stained with anti-acetylated tubulin or HNK-1 antibodies to reveal other axons in the brain, or were secondarily sectioned for light and electron microscopy. The epiphysial axons have a very specific projection pattern and virtually all axons grow precisely to their target regions without error. The first epiphysial growth cone extends ventrally from the epiphysis into the dorsoventral diencephalic tract at 19–20 h post-fertilisation (h PF). Several hours later, it turns rostrally to grow alongside axons in the tract of the postoptic commissure. The morphology of the leading growth cone changes in predictable ways at different locations along its pathway and these changes correlate with differences in the local environment that it encounters. In contrast to other published descriptions of other developing systems, the epiphysial growth cone is no more complex either when pioneering a pathway, or when encountering divergent axonal pathways. Indeed, it is most complex (i.e. has the greatest number of processes) when it first starts to follow the tract of the postoptic commissure. The presence and selective retention of filopodia within other axonal pathways suggests that growth cones have access to these pathways but do not select them. These observations support the notion that local guidance cues exist within the early scaffold of brain tracts. Subsequent epiphysial axons form a tight fascicle within the dorsoventral diencephalic tract, but abruptly defasciculate from each other upon turning rostrally into the tract of the postoptic commissure. Epiphysial growth cones that enter this tract at abnormal locations still turn in the appropriate direction. Therefore, guidance cues are not restricted solely to the normal intersections but may be distributed along the length of the tracts. The epiphysial growth cones and axons have very characteristic spatial relations to other axons in the tracts of the developing brain. They are restricted to the dorsal region of the tract of the postoptic commissure and the rostral region of the postoptic commissure. At early developmental stages, the epiphysial axons are the only axons within the dorsoventral diencephalic tract and they are located very superficially within the neuroepithelium. At later stages, they are displaced to deeper regions of the neuropil by non-epiphysial axons.