Pathfinding by pioneer neurons in isolated, opened and mesoderm-free limb buds of embryonic grasshoppers☆

Abstract The Til afferent neurons are the first neurons to undergo axonogenesis in limb buds of embryonic grasshoppers. Their growth cones pioneer a stereotyped pathway through the limb which becomes the route of one of the major leg nerve trunks. The growth cones appear to be oriented by several kinds of guidance cues, including guidepost neurons, a developing limb segment boundary, and an additional proximally orienting cue(s). In the experiments reported here, we have investigated the possible nature and source of proximally orienting and segment boundary cues by surgical manipulations of the limb. Before the onset of pioneer axonogenesis, limbs were (i) isolated from the body, (ii) opened longitudinally and pinned out flat, or (iii) stripped of mesoderm. Pioneer axon routes in cultured, surgically manipulated limb buds were compared to routes in cultured control limbs. The results indicate that proximal extension of pioneer growth cones along the limb axis does not require (during the period of growth) tissue extrinsic to the limb, contact guidance by the limb contour, an axial electrical field, a diffusion gradient generated by a localized source, mesodermal cells, or guidepost neurons; adequate guidance information for proximal growth apparently can be provided by the limb epidermal epithelium (including the basal lamina) and/or by internal polarity of the pioneer neurons. Adequate guidance information for the segment boundary portion of the pioneer route apparently can be provided by the limb epithelium.