Distribution and substrate properties of agrin, a heparan sulfate proteoglycan of developing axonal pathways

The distribution and substrate properties of agrin, an extracellular matrix heparan sulfate proteoglycan (HSPG), was investigated in the developing chick nervous system by immunocytochemistry, Western blotting, and in neurite outgrowth assays. By comparing the distribution of agrin with that of laminin-1, merosin (laminin-2), neurofilament, and neural cell adhesion molecule (NCAM), it was found that throughout development, agrin is a constituent of all basal laminae. From embryonic day (E) 4 onwards, agrin is also abundant in axonal pathways of the central nervous system, such as the optic nerve, the tectobulbar pathway, the white matter of the spinal cord, and the marginal and the molecular layers of the forebrain and the cerebellum. The abundance of agrin in brain decreases from E13 onwards. In the peripheral nervous system, agrin is present throughout development as a constituent of the Schwann cell basal laminae. Western blots confirmed the immunocytochemical data, showing maximum expression of agrin occurs during the early to medium stages of brain development. Western blots also showed that in mouse and human brain, agrin exists as an HSPG. Purified agrin did not support neurite outgrowth, rather it inhibited retinal neurite extension on mixed agrin/merosin substrates. Despite the fact that agrin, when used as a substrate inhibited neurite outgrowth, its temporal and spatial overlap with growing axons suggests that agrin has a supportive role in the development of axonal pathways, possibly as a binding component for growth factors and cell adhesion proteins.