The ability to regenerate the central nervous system (CNS) is limited to only a subset of vertebrates, such as amphibian and fish. In these robustly regenerating species, radial glial cells (RGCs) in the spinal cord have been suggested to form growth-permissive channels that precede neurite outgrowth during both regeneration and development. In the rodent spinal cord, RGCs similarly build a fibrous network of highly conserved compartments during embryonic stages when axon tracts begin to form. However, whether mammalian RGCs play similar roles in developmental longitudinal axon growth, and whether this mechanism can be harnessed to promote spinal cord regeneration, remains largely unknown. In this proposal, I will examine a novel mechanism for long distance axon growth promotion in the developing mouse spinal cord. My lab has identified a population of roof plate-derived radial glial-like cells (RGLCs) that may play an important role in the development of a major spinal cord axon tract, the dorsal column (DC). My preliminary data show that RGLCs migrate into the DC midline at embryonic day 14.5 (E14.5) as the longest DC fibers, originating from dorsal root ganglia (DRGs), simultaneously begin ascending to the medulla. By using a mouse line carrying a spontaneous mutation in Lmx1a, a gene required for the development of the roof plate, I will examine the function of RGLCs. My preliminary findings revealed that DC fibers in the mutants have a longitudinal growth deficit, and seem to ectopically cross the DC midline. I hypothesize that the RGLCs provide both physical and chemical growth support for the long distance growth promotion of DC axons, and provide a midline barrier to maintain ipsilateral segregation throughout the spinal cord.
In Aim 1, I will quantify the extent of the growth deficit in the Lmx1a mutants by using sparse genetic cell labeling in the absence of RGLCs. I will also confirm that the DC growth deficit is due to extrinsic deficiencies by comparing the intrinsic growth abilities of mutant and control DRGs. To investigate whether the protocadherin-g (PCDHG) cluster, which I found to be highly enriched in the RGLCs in my RNA seq analysis, is required for DC fiber longitudinal growth, I will analyze Pcdh-gdel/del mice for DC growth deficits. Additionally, I will examine whether the PCDHG cluster is sufficient to promote outgrowth by co-culturing transfected HEK cells with DRG explants.
In Aim 2, I will examine the extent of ectopic DC pathway midline crossings in the Lmx1a mutants using unilateral viral injections. I will also test whether Lmx1a ectopic crossings are due to absence of RGLCs or failure to respond to midline repulsion by co-culturing DRG explants with isolated RGLCs. Lastly, I will determine which guidance and inhibitory cues mediate the midline repulsion or branching inhibition of DC axons by co-culturing DRG explants with cell membranes transfected with candidate repulsive molecules from my RNAseq of RGLCs. The anticipated results of this proposal will establish a novel developmental mechanism in the spinal cord for the long distance growth and guidance of longitudinal axon tracts, and could provide insight for therapeutic approaches to mammalian spinal cord regeneration.

Public Health Relevance

Spinal cord injury can result in the permanent disruption of sensory and motor communication between the brain and periphery, but few vertebrate species have the ability to regenerate damaged axons. This study will investigate a novel mechanism for long distance axonal growth and guidance within the spinal cord during development. A better understanding of the developmental requirements for long distance growth in the spinal cord could lay a foundation for future therapeutic regenerative treatments following injury.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Riddle, Robert D
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University of Pennsylvania
Schools of Medicine
United States
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