Figure 3 Mechanisms of growth cone guidance. Growth cone guidance is modulated by four main environmental stimuli: a, chemoattraction; b, chemorepulsion, mediated by soluble molecules; c, contact attraction; and d, contact repulsion, mediated by substrate-bound molecules.

time when axons meet with its region of expression; (2) it must be able to steer axons in vitro and in vivo; and (3) interfering with its function should result in axon guidance and targeting errors. The recent advent of powerful molecular and genetic techniques enabled these criteria to be met:

1. Analysis of the expression patterns of the protein of interest can be performed using antibody staining, application of tagged protein probes, or of RNA by in situ hybridization, screening through different developmental stages and target tissues.

2. To test for guidance activity in vitro, several experimental paradigms have been developed. One of them is called the stripe assay (Figure 4a). In this assay, axons are given a choice of growing on alternating lanes of guidance molecule versus control protein. If the guidance molecule tested is an attractive one, the axons will grow on the lanes containing the guidance molecule. If the guidance molecule tested acts as a repellent, then axons will avoid the lanes containing the guidance molecule and will grow in the control protein lanes.

Other in vitro guidance tests are also common. The growth cone collapse assay is a chemotropic assay for repulsive guidance cues, in which the protein of interest is applied globally to the growing axons for a certain amount of time (Raper and Kapfhammer, 1990). After application of the protein of interest, the percentage of collapsed growth cones is counted. Collapse is defined as a loss of the motile structures of the growth cone (the lamellipodia and the filopodia) and is often accompanied by cessation of growth and withdrawal of the growth cone (Figure 4b). Collapse in vitro is thought to be correlated with repulsion in vivo. An assay to test the guidance activity of substrate-adhered proteins, the bead assay, is also widely used. In this assay, a protein-coated latex bead is manipulated with the help of a laser tweezer (Kuhn et al, 1995; Gallo et al., 1997). The advantages of this system are that the concentration of the protein on the bead surface can be regulated, the beads can be immobilized accurately at the desired position, and the presentation on the bead surface may mimic the

Stripe assay

Stripe assay

(a) Control

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