Axonal Growth on Solubilized and Reconstituted Matrix from the Embryonic Chicken Retina Inner Limiting Membrane

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Abstract
Basal laminae, thin sheets of extracellular matrix covering the basal side of all neuroepithelia, are strongly supportive for neurite outgrowth in vitro and may provide a permissive environment for growing neurites in vivo. To gain information about the biological activity and composition of in situ-derived basal laminae the inner limiting membranes from embryonic day (E) 7 to E11 chick and quail retinae were isolated. The basal laminae were solubilized with high-molar guanidine hydrochloride or urea, and the solubilized proteins reconstituted by dialysis. The matrix proteins were spotted or dried onto nitrocellulose or polylysine-coated dishes. When explants from retina or from dorsal root ganglia were incubated on the protein spots, neurite extension was very robust, at a level as high as on authentic basal lamina. Extracts from the pigment epithelial basement membrane did not support neurite extension. Western blot analysis showed that the explant from the retinal inner limiting membrane contained predominantly basal lamina-type proteins, such as laminin, collagen type IV and heparan sulphate proteoglycan, whereas the matrix extract from the pigment epithelium contained predominantly mesenchymal-type proteins, like collagen type I and tenascin. JG22, a beta1 integrin antibody that inhibited neurite extension on EHS tumour laminin substrate, had no effect on neurite outgrowth on retinal basal lamina matrix, indicating that embryonic basal laminae contain other or additional growth promoting substrate molecules.