Functional Analysis of Candidate Genes Identified from Naturally Regenerating Spinal Cords of the Salamanser (Ambystoma)

Salamanders are promising models for spinal cord injury (SCI) because they are the only vertebrates capable of complex organ regeneration in the adult phase. Unfortunately, little is known about mechanisms that allow salamanders to naturally regenerate complex organs such as spinal cord, and how these mechanisms relate to mammalian models of SCI. Previous KSCHIRT funding supported development of the first salamander Affymetrix GeneChip. We used this new resource to profile early gene expression during spinal cord regeneration (SCR) and to identify new candidate genes that are upregulated uniquely in salamander. Now that we have identified SCR candidate genes, a logical next step is to develop and apply methods that will allow the function of these genes to be assayed in the salamander system. Characterization of candidate gene functions in the salamander system is needed to rovide basic understandin of re enerative mechanisms and to effectivel rioritize enes for future studies in mammalian SCI models. Thus we ro ose in S ecific Aim 1 to knock-down ex ression of candidate genes in vivo that are up-regulated during SCR in salamander. Recent studies show that morpholino anti-sense oligonucleotides offer a reliable and robust methodology for knocking-down in vivo translation in Ambystoma. We propose to design morpholinos for matrix metalloproteinase (Mmp) and Wnt family members and electroporate them into ependymal cells that line the lumen of the spinal cord. We hypothesize that Mmp and Wnt family candidate genes are up-regulated during SCR because they function in signaling pathways that regulate ependymal cell activation and proliferation. If this hypothesis is correct, then our knock-down studies promise to identify signaling pathways that can be manipulated to initiate neurogenesis in mammalian ependymal cells after SCI. Although gene knockdown studies will greatly extend the salamander system in SCI research, many additional ependymal cell biomarkers and an ependymal cell line are needed. Thus, we propose in Specific Aim 2 to isolate e end mal cells from re eneratin adult sinal cord to identi new ene candidates and establish the first long-term axolotl ependymal cell line for in vitro studies. Accomplishment of these specific aims will extend the salamander model system in SCI research. VI. Relationship to Kentucky Spinal Cord and Head Injury Research Board priorities: The proposed research will identify genes and gene pathways that are critical for enhancing regeneration after spinal cord injury. The project will leverage current and continuing investment in salamander biology and genome resource development by NIH, NSF and the University of Kentucky. Although the PI has established a successful program in genome research and resource development (NIH R24 mechanism), KSCHIRT funding is needed to generate additional background data and molecular resources to ensure competitiveness for SCI funding via the NIH RO1 mechanism. 4