Ultrastructural observations on the expression of axonin-1: Implications for the fasciculation of sensory axons during axonal outgrowth into the chick hindlimb

To help understand how axons interact as they grow into the developing chic k hindlimb, we used electron microscopy in conjunction with immunoperoxidas e staining for the cell adhesion molecule axonin-1 to label sensory axons. The results showed that sensory axone travel together in bundles, tightly a pposed to one another. In contrast, motoneuron axons are more widely spaced , although motoneuron axons situated at the perimeter of sensory axon bundl es are found in close contact with neighboring sensory axons. Sensory growt h cones and lamellipodia tend to be located centrally within the bundles, w ith several lamellipodia typically being found stacked together. Strikingly , regions of close axonal apposition are accompanied by axonin-1 expression , suggesting that such contacts are indeed adhesive. Taken together, these observations suggest that groups of sensory axons of a similar age grow tog ether, with some of the older sensory axons fasciculating along motoneuron axons and younger sensory axons later fasciculating along older sensory axo ns. Axons situated at the periphery of sensory bundles are typically partly lab elled, such that axonin-1 is expressed on membranes apposing other labelled axons but not on those facing unlabelled axons or unlabelled Schwann cells . Thus, axonin-1 appears to become redistributed within the membranes of ax ons growing into the limb, as it does on cultured neurons. In contrast, the neuron-glia cell adhesion molecule (NgCAM), which binds heterophilically t o axonin-1, appears uniformly distributed on even those axons that would ha ve an asymmetric distribution of axonin-1. Thus, the localization of axonin -1 strongly suggests that it plays an important role in sensory axon fascic ulation, but the relative contributions of its interactions with various po tential ligands are unclear. Finally, we found that some sensory growth cones have lamellipodia that are spread over considerable expanses. This suggests that although fasciculati on is important in sensory axon guidance, sensory axons may also explore th e local environment. (C) 1999 Wiley-Liss, Inc.