Clustering transmembrane-agrin induces filopodia-like processes on axons and dendrites.

The transmembrane form of agrin (TM-agrin) is primarily expressed in the CNS, particularly on neurites. To analyze its function, we clustered TM-agrin on neurons using anti-agrin antibodies. On axons from the chick CNS and PNS as well as on axons and dendrites from mouse hippocampal neurons anti-agrin antibodies induced the dose- and time-dependent formation of numerous filopodia-like processes. The processes appeared within minutes after antibody addition and contained a complex cytoskeleton. Formation of processes required calcium, could be inhibited by cytochalasine D, but was not influenced by staurosporine, heparin or pervanadate. Time-lapse video microscopy revealed that the processes were dynamic and extended laterally along the entire length of the neuron. The lateral processes had growth cones at their tips that initially adhered to the substrate, but subsequently collapsed and were retracted. These data provide the first evidence for a specific role of TM-agrin in shaping the cytoskeleton of neurites in the developing nervous system.

On the turning of Xenopus retinal axons induced by ephrin-A5.

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, play important roles during development of the nervous system. Frequently they exert their functions through a repellent mechanism, so that, for example, an axon expressing an Eph receptor does not invade a territory in which an ephrin is expressed. Eph receptor activation requires membrane-associated ligands. This feature discriminates ephrins from other molecules sculpturing the nervous system such as netrins, slits and class 3 semaphorins, which are secreted molecules. While the ability of secreted molecules to guide axons, i.e. to change their growth direction, is well established in vitro, little is known about this for the membrane-bound ephrins. Here we set out to investigate--using Xenopus laevis retinal axons--the properties of substratum-bound and (artificially) soluble forms of ephrin-A5 (ephrin-A5-Fc) to guide axons. We find--as expected on the basis of chick experiments - that, when immobilised in the stripe assay, ephrin-A5 has a repellent effect such that retinal axons avoid ephrin-A5-Fc-containing lanes. Also, retinal axons react with repulsive turning or growth cone collapse when confronted with ephrin-A5-Fc bound to beads. However, when added in soluble form to the medium, ephrin-A5 induces growth cone collapse, comparable to data from chick. The analysis of growth cone behaviour in a gradient of soluble ephrin-A5 in the 'turning assay' revealed a substratum-dependent reaction of Xenopus retinal axons. On fibronectin, we observed a repulsive response, with the turning of growth cones away from higher concentrations of ephrin-A5. On laminin, retinal axons turned towards higher concentrations, indicating an attractive effect. In both cases the turning response occurred at a high background level of growth cone collapse. In sum, our data indicate that ephrin-As are able to guide axons in immobilised bound form as well as in the form of soluble molecules. To what degree this type of guidance is relevant for the in vivo situation remains to be shown.