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Spinal Cord Regeneration in the Leopard Gecko (Eublepharis macularius): Investigating the Stemness, Activation and Heterogeneity of Ependymal Layer Cells

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Title: Spinal Cord Regeneration in the Leopard Gecko (Eublepharis macularius): Investigating the Stemness, Activation and Heterogeneity of Ependymal Layer Cells
Author: Gilbert, Emily Anne Beatrice
Department: Department of Biomedical Sciences
Program: Biomedical Sciences
Advisor: Vickaryous, Matthew
Abstract: The leopard gecko (Eublepharis macularius) is an emerging model for the study of spinal cord regeneration. As for many lizards, geckos are able to self-detach their tail to avoid predation and then regenerate a replacement. The replacement tail includes a regenerated spinal cord with a relatively simple morphology: a monolayer of ependymal layer cells (ELCs) surrounded by nerve tracts. We hypothesized that ELCs of the original spinal cord include populations of neural stem/progenitor cells (NSPCs) that contribute to the fully regenerated spinal cord. To identify ELCs as NSPCs, we first used a bromodeoxyuridine pulse-chase experiment to document label-retaining populations prior to injury. We found that populations of ELCs were label-retaining, even after a 140-day chase period. Next, we conducted a detailed spatiotemporal characterization of ELCs before, during, and after tail regeneration. We determined that ELCs are activated in response to injury, as evidenced by changes in proliferation and protein expression. Prior to injury, ELCs express the hallmark NSPC marker SOX2. Following tail loss, ELCs in the original stump of the tail become highly proliferative and express an expanded panel of NSPC and lineage-restricted progenitor cell markers, including Musashi-1, SOX9, β-III tubulin and HuC/D. This panel of markers continues to be expressed by ELCs of the fully regenerated tail, and indicates that the replacement spinal cord includes at least two distinct populations. Most ELCs are SOX2+, GFAP+, HuC/D-. We interpret these cells as ependymo-radial glia (ERG). A second subset of ELCs are SOX2+, GFAP-, HuC/D+, which we identify as a neuronal-like population of cerebrospinal fluid-contacting (CSF-c) cells. Although we were unable to successfully track labeled ELCs into the regenerating tail following in vivo electroporation, our data provides compelling evidence that this cell population is crucial for restoring a functional spinal cord. Our in vitro experiments indicate that culturing methods common to mammalian neural tissues are not necessarily well-suited to reptiles. These studies serve as a foundation for future experimental work that promises to advance our understanding of the biology of spinal cord repair and regeneration, with obvious implications for biomedicine and translational science.
Date: 2016-09
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