Isolation, characterization, and substrate properties of the external limiting membrane from the avian embryonic optic tectum

The external limiting membrane of the avian embryonic optic tectum
is isolated by mechanically separating the neuronal mesencephalon
from the overlying mesenchymal tissue. The preparation consists of
a basal lamina which is covered on its neural side by endfeet of
neuroepithelial cells and has attached to it on its meningeal side
a collageneous stroma, containing blood vessels. The external
limiting membrane can be flat-mounted on a piece of nitrocellulose
filter as mechanical support. It covers an area between 0.3 and 1
the cm2, depending on the age of me donor embryo. The endfeet can
be removed together with all cellular components of the meninges by
treatment with 2% Triton-X-100 or with distilled water. The basal
lamina itself is approximately 80 nm thick and consists of two
laminae rarae and a central lamina densa. Immunohistochemical
staining reveals that the basal lamina in the embryo, after
isolation and after detergent extraction of the isolated
preparation, contains type IV collagen, nidogen, laminin, and low
density heparan sulfate proteoglycan as do other basement
membranes. Antibodies against the neural cell adhesion molecule
(N-CAM), chondroitin sulfate proteoglycan, and fibronectin fail to
stain the external limiting membrane, but these proteins were
clearly identified in the blood vessel-containing meninges or in
the optic tectum. The flat-mounted external limiting membrane
preparation was used as substrate to culture several different
neural tissues of central and peripheral origin. Explants of neural
crest cells, dorsal root ganglia, and sympathetic ganglia can be
cultured on the external limiting membrane. All explants grow well
on the basal lamina preparations whether the endfeet are attached
or detergent-extracted prior to explantation; however, neurite
outgrowth from sympathetic ganglia is reduced in the presence of
the endfeet. Although the endfoot-lined external limiting membrane
represents at least part of the immediate environment encountered
by retinal axons as they invade the optic tectum and despite its
excellent properties as a substrate for retinal axons in vitro,
cues guiding the orientation of axons were not detected in the
flat-mounted preparation.