Removal of the basal lamina in vivo reveals growth cone-basal lamina adhesive interactions and axonal tension in grasshopper embryos

Abstract

The Ti1 afferent neurons are the first cells to undergo axonogenesis in embryonic grasshopper limbs. The Ti1 growth cones migrate between the limb epithelium and its basal lamina. We have investigated the nature of growth conebasal lamina interactions in vivo by removing the basal lamina with mild enzymatic digestion. Treatment with elastase, ficin, or papain removes the basal lamina when viewed in scanning electron microscopy. Trypsin and chymotrypsin leave the basal lamina intact. If the basal lamina is removed after the Ti1 growth cones have extended over intrasegmental epithelium but are not yet in contact with either differentiated segment boundaries or neurons, the growth cones retract to the cell somata. If the basal lamina is removed by elastase, and the Ti1 neurons are allowed to extend axons after treatment, a second elastase digestion does not cause the axons to retract. It is therefore unlikely that axon retraction is due to general proteolysis. These results suggest that if Ti1 growth cones have initially extended in the presence of an intact basal lamina, they are dependent on the lamina to remain extended over this region of the limb. The retraction of the Ti1 axons after removal of the basal lamina is inhibited by cytochalasin D, suggesting that microfilament-based cytoskeletal components underlie this event. This result indicates that the axons are under tension in vivo. The ability of the Ti1 growth cones to resist axonal tension suggests that adhesive interactions between the growth cones and the basal lamina underlie normal axon outgrowth in vivo.