Response of retinal ganglion cell axons to striped linear gradients of repellent guidance molecules

Abstract

Although molecular gradients have long been postulated to play a role in the development of topographic projections in the nervous system, relatively little is known about how axons evaluate gradients. Do growth cones respond to concentration or to slope? Do they react suddenly or gradually? Is there adaptation? In the developing retinotectal system, temporal retinal ganglion cell axons have previously been shown to avoid repellent cell-surface activities distributed in gradients across the optic tectum. We confronted temporal retinal axons with precisely formed striped linear gradients of repellent tectal membranes and of two candidate repellent molecules, ephrin-A2 and -A5. Axons entered gradient stripes independently of their slope and extended unhindered in the uphill direction until they suddenly avoided an apparent threshold concentration of repellent material that was independent of slope. This critical concentration was similar in both linear and nonlinear gradients, and hence independent of gradient shape. When gradients of identical slope were formed on different basal levels of repellent material, axons grew uphill for a fixed increment of concentration, possibly measured from the lowest point of the gradient, rather than up to a fixed absolute concentration. The speed of growth cones was not affected by repellent unstriped gradients below the critical concentration level. Similar results were found with membranes from cell lines stably transfected with either ephrin-A5 or ephrin-A2, two previously identified growth cone repellent cell-surface proteins. These data suggest that growth cones or axons can integrate guidance information over large distances, probably by a combined memory and adaptation mechanism.

Reading of concentration gradients by axonal growth cones

“…In these versions of gradient models target position is dependent on absolute concentrations of guiding molecules, not on slope. Thus the gradient would give two commands to the growth cone, for example, (i) grow uphill, and (ii) stop at the concentration c. The latter view has recently been corroborated by experiments of Rosentreter et al (1998). They showed in in vitro gradient assays, that temporal axons react to the cue at a de¢ned concentration within the gradient irrespective of gradient slope.…”

“…(d) A step-like behaviour in the gradient assay remains di¤cult to explain In order to investigate axons growing on graded substrates, we have developed an in vitro assay (Rosentreter et al 1998) in which the substrate is o¡ered as a linear concentration gradient of guidance molecules. This has been done in both striped and non-striped gradients.…”

“…Like the stripe assay, the gradient assay reveals temporal and nasal behaviours but no intermediate behaviour (Rosentreter et al 1998). All temporal axons are shorter than the nasal axons.…”

“…However, there is some evidence for receptor adaptation in the gradient assay (Rosentreter et al 1998).…”

“…Imprint matching in this model is due to the fact that the cis-ligands induce a desensitization of their bound receptors, thereby determining the concentration of remaining sensitive receptors. In addition the model is also able to provide an explanation of the in vitro gradient experiments by Rosentreter et al (1998) described in ½ 5(c). As long as the growth cone migrates on a homogeneous substrate the di¡erential sensor for the spatial inhomogeneity of the guidance signal would be silent, thereby permitting desensitization of the signalling receptors.…”