The avian tectobulbar tract: development, explant culture, and effects of antibodies on the pattern of neurite outgrowth

The tectobulbar tract is the first long-distance projecting fiber
pathway to appear during the development of the avian optic tectum
(dorsal half of the mesencephalon). Immunologically stained
wholemounts of the E3 mesencephalon reveal that the early
tectobulbar axons course in a dorsal-to-ventral direction and
abruptly turn in a caudal direction shortly before reaching the
ventral midline. During subsequent development, more tectobulbar
axons are generated that form a parallel array of thick fascicles
coursing ventrally within the mesencephalon. At this later stage of
development, the tectobulbar tract bifurcates into an ipsilateral
and contralateral pathway, both growing in a caudal direction near
the mesencephalic ventral midline. Bifurcation and change in
direction of growth is accompanied by a complete loss of the
fasciculated growth pattern. Each tectobulbar axon is thus divided
into a proximal fasciculated and a distal unfasciculated segment.
Tectobulbar fascicles occupy the most superficial surface layer of
the mesencephalon at early stages and are displaced deeper into the
tissue beginning at embryonic day 5. In both of these locations,
tectobulbar axons express molecules involved in axon-axon and
axon-substrate interactions like the G4 antigen, neural cell
adhesion molecule (N-CAM), neurofascin, and T61 antigen as revealed
by immunohistochemistry and immunoblotting. Stripes of the
mesencephalon explanted onto a basal lamina substratum show
vigorous outgrowth of neurites. These processes grow in fascicles
at a growth rate of 40 microns/h. Staining of the neurites with
specific antibodies, as well as the position of the retrogradely
labeled cell bodies, is in agreement with these processes being
tectobulbar axons. This in vitro explant system was used to
investigate the expression and possible functional involvement of
N-CAM, neurofascin, G4 protein, and T61 antigen in the growth of
these axons. The presence of antigen- binding fragments of
polyclonal anti-G4 antibodies completely blocks fasciculation of
the neurites but has no influence on their rate of elongation.
Antibodies against N-CAM and neurofascin have no detectable
effects. The number and length of the in vitro growing axons are
reduced by the monoclonal T61 antibody. This effect is reversible.
The elucidation of the exact course in vivo and the accessibility
to the axons growing in vitro make the tectobulbar tract an
excellent model system for the investigation of the role of these
and other proteins in axonal growth and guidance during the
development of the CNS.