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

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 cm2, depending on the age of the 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.