PRECISELY CONTROLLED GRADIENTS FOR AXON GUIDANCE

Georgetown University, Washington DC

Investigators

Abstract

DESCRIPTION (Verbatim from the Applicant's Abstract): As the brain develops, axons often grow over long distances to find appropriate targets. Some of the most important guidance cues they use to achieve this feat are concentration gradients of chemotropic molecules. These gradients are detected by the growth cone, a complex and sensitive structure at the tip of the developing axon. Understanding how growth cones detect gradients is crucial for understanding how the brain normally develops, how defects in normal wiring can occur, and how axons can appropriately regenerate after injury. However, there is currently very little quantitative information about how growth cones respond to gradients. This is primarily due to the absence of an experimental technique for establishing gradients of diffusible axon guidance molecules in vitro, such that the shape and steepness of the gradient is under the control of the experimenter. Without such a technique it is impossible to systematically vary the parameters of the gradient, and thus impossible to determine quantitatively how each of these parameters regulates axon guidance. The goal of this project is to develop and implement a novel technique to make these measurements possible. We will establish gradients by "printing" chemotropic molecules onto the surface of a long, thin block of collagen gel in which axons are growing, and allowing the molecules to diffuse into the collagen. Precisely controlled volumes of chemotropic factor can be deposited at precisely controlled locations using a method similar to that employed in inkjet printers. Because molecular movement by diffusion is fast over short distances but slow over long distances, initial local unevenness in the concentration smoothes out in a few hours, leaving behind a gradient that remains smooth and stable for several weeks. We thus have complete control over the shape of this gradient via control over the pattern of molecules deposited on the surface. By observing axon growth and turning behavior in these gradients we will obtain quantitative data of unprecedented precision on the parameters controlling axon guidance by diffusible gradients. Our initial model system will be the guidance of rat dorsal root ganglion axons by gradients of Nerve Growth Factor. However, we expect this technology to be also widely applicable to other model systems for axon guidance, and more generally to studies of cell motility and chemotaxis.