University of Kentucky
Localized Gene Expression of Guidance Molecules in a Co-Culture Model to Direct Axonal Growth
Institution
University of Kentucky
Faculty Advisor/ Mentor
Diane Snow; George Smith
Abstract
Axonal growth cones are guided to their respective target locations by contact-dependent mechanisms or by diffusible long-range chemotropic factors. The identification of chemoattractive and chemorepulsive guidance molecules is essential for promoting axon regeneration following neuronal injury in vivo. Three dimensional collagen gel assays are commonly used to study axon guidance by diffusible factors. Alternatively, contact dependent axon guidance is routinely tested by monitoring growth cone responses to substratum-adsorbed molecules. Here we have developed two, novel, more physiologically relevant methods in vitro to create patterned gene expression of guidance molecules in a complex cellular environment with the goal of more closely mimicking axon outgrowth in vivo. To examine the usefulness of these techniques for axon guidance, the growth response of axons from chicken dorsal root ganglia explants was studied in both culture systems. These paradigms demonstrated regulation of neurite outgrowth in response to the cell surface, inhibitory chondroitin sulfate proteoglycan, Brevican, and to a secreted repulsive glycoprotein, Sema 3A. From analyses of neurite behavior, we conclude that the two methods can be used to generate expression patterns of growth-regulatory proteins in a complex cellular environment. Further, we validated that not only cell surface molecules can be accurately presented, but also soluble glycoproteins, by maintaining the cultures in Matrigel® to limit diffusion away from the infected cells. The ultimate goal of such assays will be to develop pharmacotherapeutic strategies to facilitate growth and regeneration of nerve cells in order to promote regeneration and recovery of function in the injured adult.
Localized Gene Expression of Guidance Molecules in a Co-Culture Model to Direct Axonal Growth
Axonal growth cones are guided to their respective target locations by contact-dependent mechanisms or by diffusible long-range chemotropic factors. The identification of chemoattractive and chemorepulsive guidance molecules is essential for promoting axon regeneration following neuronal injury in vivo. Three dimensional collagen gel assays are commonly used to study axon guidance by diffusible factors. Alternatively, contact dependent axon guidance is routinely tested by monitoring growth cone responses to substratum-adsorbed molecules. Here we have developed two, novel, more physiologically relevant methods in vitro to create patterned gene expression of guidance molecules in a complex cellular environment with the goal of more closely mimicking axon outgrowth in vivo. To examine the usefulness of these techniques for axon guidance, the growth response of axons from chicken dorsal root ganglia explants was studied in both culture systems. These paradigms demonstrated regulation of neurite outgrowth in response to the cell surface, inhibitory chondroitin sulfate proteoglycan, Brevican, and to a secreted repulsive glycoprotein, Sema 3A. From analyses of neurite behavior, we conclude that the two methods can be used to generate expression patterns of growth-regulatory proteins in a complex cellular environment. Further, we validated that not only cell surface molecules can be accurately presented, but also soluble glycoproteins, by maintaining the cultures in Matrigel® to limit diffusion away from the infected cells. The ultimate goal of such assays will be to develop pharmacotherapeutic strategies to facilitate growth and regeneration of nerve cells in order to promote regeneration and recovery of function in the injured adult.