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. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 541–562, 1998

Multi-Molecular Gradients of Permissive and Inhibitory Cues Direct Neurite Outgrowth

“…4,5,19,33,38 We analyzed our data from single-cue gradient experiments in the context of the two models of gradient detection: absolute change (to which we refer as slope) and fractional change in concentration. Within our system, the molecular concentration varied linearly across the 250 lm width of each gradient channel, as confirmed by immunofluorescence.…”

“…14,15,25 Experimental work has suggested that axons could be guided by a wide range of parameters including the absolute molecular concentration, the gradient sign or direction, the gradient shape, the gradient slope or steepness, and the fractional change in concentration. 3,16,19,33,38,43 Previous studies of axon guidance by gradients have largely focused on examining diffusible gradients of soluble molecules such as neurotrophic factors, semaphorins, and netrins. 2,3,21,27,38 Recently, gradients of soluble cues have been deposited on the surfaces of three-dimensional collagen gels.…”

“…21,27 Thus, it is important to consider the direction of axon growth by surface-bound gradients, including gradients of soluble molecules, extracellular matrix molecules, and cell surface molecules. While axon guidance by discontinuous surface-bound molecules has been characterized for a number of systems, typically using variations of the stripe assay, 33,37 until recently it has been difficult to generate reproducible continuous gradients of surface-bound molecules in vitro. Recent studies have demonstrated the fabrication of molecular gradients with high degrees of resolution and control over gradient parameters through microfluidic techniques, achieving definition on the scale of hundreds of microns.…”

Getting into shape: Optimal ligand gradients for axonal guidance

“…In other words, if the axon has sufficiently explored the local environment and can no longer lower d, it may decide to stop at its current position. This proposition may be corroborated by empirical evidence showing that axons tend to stop at a defined ephrin concentration regardless of the slope of the ligand gradient (Rosentreter et al, 1998). Thus, d may itself constitute a second stop signal used by the axon.…”

The development of retinotectal maps: A review of models based on molecular gradients

“…While nasal axon growth was not affected by the gradient in this assay, temporal axons grew an amount that was negatively correlated with the steepness of the gradient (i.e., the steeper the gradient, the shorter the axons). Using a more refined version of this technique, Rosentreter et al (1998) then suggested that temporal axons grow up a gradient of posterior tectal membranes until they have travelled a fixed increment of concentration that is independent of both the slope of the gradient and the starting concentration. However, this conclusion relies on strong quantitative inferences drawn from somewhat qualitative criteria for assessing the stopping point of the front of axons within a lane.…”