Pioneer growth cone morphologies reveal proximal increases in substrate affinity within leg segments of grasshopper embryos

Abstract

We have compared the morphologies of approximately 5000 antibody-labeled afferent pioneer growth cones fixed at various stages of growth along their characteristic path over the epithelium in the legs of grasshopper embryos, and have used growth cone morphology as an indicator of differences in the affinity of the epithelial substrate for pioneer growth cones in vivo. Growth cone morphologies differ markedly between different locations in limb buds, and also in the same location in limbs at different stages of differentiation. Growth cones characteristically extend branches and lamellae circumferentially along segment boundaries, and filopodia and lamellae are retained (or extended) longer there. Where they contact a relatively well-differentiated segment boundary, the growth cones also abruptly reorient circumferentially. In the proximal regions of limb segments, growth cones consistently have a high degree of branching and lamellae; previously formed axons also extend secondary branches and spread there as development progresses. Low incidence of these morphologies is observed at all stages in the distal regions of limb segments. Thus, neuronal morphologies correlate both spatially and temporally with the differentiation of limb segmentation. These results suggest the following: Detailed growth cone morphology is a reliable indicator of differences in extrinsic guidance cues. The affinity of the epithelial substrate for afferent pioneer growth cones increases proximally within segments, with a peak at the segment boundary. (This affinity could be based on surface density of adhesion molecules or on nonadhesive molecules that actively regulate growth cone extension.) Increasing epithelial affinity within segments appears to act as a proximal guidance cue for afferent pioneer growth cones. Pioneer growth cones are observed to navigate proximally in circumstances where proximally located guidepost cells differentiate too late to guide them.