Morphogenetic guidance cues can interact synergistically and hierarchically in steering nerve cell growth

Nerve cell growth is influenced by guiding properties of its substratum. Microfabricated cell culture substrata were used to determine whether rat dorsal root ganglia (DRG) nerve cells could detect and integrate simultaneous model adhesive and topographic guidance cues. Interference reflection microscopy demonstrated strips of surface contact under the marginal zone of growth cones on planar surfaces which were coincident with actin immunostaining at the periphery of the C-domain. Clusters of focal contacts below the growth cone C-domain delineated the track edges on adhesive gratings. Neurite extension was guided most effectively by adhesive gratings of 25-µm period where highly aligned cells were typically bipolar. Nanometric steps and differences in surface texture between the adhesive tracks was detected using atomic force microscopy (AFM). Neurites did not align to 12-to l00-µm pitch grooves which were less than 1 µm deep. The proportion of aligned neurites increased with groove depth. Maximum neurite alignment was seen when 6-.im-deep, 25-11m-wide grooves contained superimposed parallel adhesive tracks of matched pitch. Neurites aligned preferentially to adhesive tracks superimposed orthogonally over shallow grooves (1 µm deep). Primary neurites aligned increasingly to grooves with orthogonal adhesive tracks as their depth increased. These neurites frequently had highly branched terminal arbours aligned to the orthogonal adhesive tracks. We conclude that morphogenetic guidance cues can interact synergistically and hierarchically to steer nerve cell growth.