The mechanisms by which neuronal pathways are established are not well understood. One theory states that pathways are first laid out by pioneer neurons and that subsequent axons travel along them. This pattern has yet to be demonstrated in the vertebrate CNS. I will test this theory by examining development of descending tracts in the relatively simple caudal spinal cord of embryos and tadpoles of Xenopus laevis. First, I will use light and electronmicroscopy to determine the time of appearance and location of the earliest tract axons. I will then identify cells giving rise to them using retrograde transport of HRP from sites near the caudal tip of the spinal cord. Axons which enter the tracts later will be examined using light and electron microscopy and HRP backfilling from progressively more rostral levels. Once the earliest neurons have been identified, I will selectively delete them by backfilling with Lucifer yellow, a fluorescent dye which when exposed to blue light destroys cells containing it. My preliminary studies show that tracts develop by ingrowth of axons into preexisting spaces between adjacent neuroepithelial cells. When the caudal-most front of tract growth is compared with progressively more rostral levels it can be seen that spaces and, later, fascicles, appear in consistent sequence and position. HRP backfills of the most primitive tract level show that primary sensory and motor cells initiate the two earliest fascicles, one dorso- and one ventrolateral. Filling at more rostral levels shows that additional primary neurons are added before spinal interneurons and that axons from trunk and supraspinal levels appear only after many axons of local origin are already present. These pilot data suggest that in descending tracts local primary neurons may first guide each other in overlapping segmental arrangement and, later, guide spinal and supraspinal interneurones. Primary neurons may thus serve as """"""""segmental pioneers."""""""" The proposed experiments will further define the components and sequence of tract development and test an important theory of axonal guidance. The paradigm developed for these studies can be applied in the future to comparison of normal and regenerative spinal cord development.
