Filopodial adhesion does not predict growth cone steering events in vivo

Migration of growth cones is in part mediated by adhesive interactions betw een filopodia and the extracellular environment, transmitting forces and si gnals necessary for pathfinding. To elucidate the role of substrate adhesiv ity in growth cone pathfinding, we developed an in vivo assay for measuring filopodial-substrate adhesivity using the well-characterized Ti pioneer ne uron pathway of the embryonic grasshopper limb. Using time-lapse imaging an d a combination of rhodamine-phalloidin injections and Dil labeling, we dem onstrate that the filopodial retraction rate after treatment with cytochala sin D or elastase reflects the degree of filopodial-substrate adhesivity. M easurements of filopodial retraction rates along regions of known differing substrate adhesivities confirmed the use of this assay to examine filopodi al-substrate adhesion during in vivo pathfinding events. We analyzed 359 fi lopodia from 22 Ti growth cones and found that there is no difference betwe en the retraction rates of filopodia extending toward the correct target (o n-axis) and filopodia extending away from the correct target (off-axis). Th ese results indicate on-axis and off-axis filopodia have similar substrate adherence. Interestingly, we observed a 300% increase in the extension rate s of on-axis filopodia during Ti growth cone turning events. Therefore, in addition to providing filopodia with important guidance information, region al cues are capable of modulating the filopodial extension rate. The homoge neity in filopodial retraction rates, even among these turning growth cones in which differential adhesivity might be expected to be greatest, strongl y establishes that differential adhesion does not govern Ti pioneer neuron migration rate or pathfinding. We propose that the presence of local differ ences in receptor-mediated second messenger cascades and the resulting asse mbly of force-generating machinery may underlie the ability of filopodial c ontacts to regulate growth cone steering in vivo.