Neural Stem Cell Properties and Amphibian Spinal Cord Regeneration

K. Sato, A. Corn, R. Karcavich, R. C. Smith* and E.A.G. Chernoff

IUPUI Center for Regenerative Biology and Medicine
Eli Lilly and Co.* Indianapolis, IN 46202

      Functional recovery of lesioned vertebrate spinal cord can involve axonal regrowth or reconstruction with neurogenesis. The driving forces for spontaneous spinal cord regeneration involve retention of “embryonic” properties, reconstruction of an “embryonic” environment and the intrinsic ability of neurons and glia to respond to such an environment. One component of this process is the activation of neural stem cells following injury.

      Xenopus laevis (clawed frog) spinal cord regenerates when lesioned in stage 50 tadpoles, but fails to regenerate in tadpoles lesioned at stage 62. Proliferating cell nuclear antigen (PCNA) localization in stage 50 tadpoles shows that the ependymal cells (ventricular zone glia) within the regenerating tadpole spinal cord are highly mitotic and contribute to the regeneration outgrowth. A subtractive screen between stage 50 and stage 62 cord shows that several Notch-1 signaling pathway members are differentially expressed in cord at stage 50. The downstream effecter of Notch, Enhancer of Split-Related protein (ESR-7) and the Notch ligand Delta-2 are identified in the Stage 50 minus Stage 62 subtraction. In situ hybridization shows that ESR-7 and Delta-2 are expressed in ependymal cells. Other members of the Notch pathway were examined including the neural progenitor and stem cell marker Nrp-1, the Xenopus homolog of Musashi-1. Musashi-1 expression inhibits Numb translation and maintains notch signalling. Nrp-1 is strongly expressed in regeneration competent Xenopus spinal cord. The Nrp-1 mRNA is localized in ependymal cells and dorsal subependymal cells in cord and in forebrain. An inhibitor of neuronal differentiation outside of the Notch pathway, Id3 (Inhibitor of DNA-binding) is also expressed in ependymal cells. Id3 has been described by others in association with stem cell properties in other tissues and organisms. A Xenopus tadpole ependymal cell culture model system was devised to permit experimental manipulation of these cells. The stage 50-derived ependymal cells express Nrp-1/Musashi-1 in culture. Like late embryonic or adult mammalian neural stem cells, the ependymal cells respond to EGF or FGF-2 in culture, producing neurospheres. EGF and FGF-2 also act synergistically. The in vivo and in vitro expression of genes associated with stem cell properties, and neurosphere formation in culture, suggests that Xenopus spinal cord regeneration competence may be correlated with neural stem cell behavior of the ependymal cells. Supported by the NSF, Eli Lilly and Co. and the Indiana 21st Century Research and Technology Fund.