Transcriptomic analysis identifies injury-responsive fibroblast populations as potential mediators of Wnt-dependent spinal cord regeneration

In humans and other mammals, spinal cord injury (SCI) can lead to a permanent loss of sensory and motor function, due to the inability of damaged neurons and axons to regenerate. However, other vertebrate species including zebrafish exhibit complete spinal cord regeneration and functional recovery after SCI. Wnt signaling is required for neurogenesis and axon regrowth in a larval zebrafish SCI model, but the genes regulated by this pathway and the cell types that express them remain largely unknown. In this study, we used bulk RNA-sequencing (RNAseq) to identify candidate genes regulated by Wnt signaling that are expressed after SCI. Using this unbiased screen, we identified multiple genes previously unassociated with SCI in larval zebrafish. Our data reveal potential novel gene targets and cell populations that may play important roles in spinal cord regeneration. Wild Type larval (AB) zebrafish were subjected to full mechanical transection of the spinal cord at the level of the anal pore at 5 days post fertilization. After a 6hr recovery period, larvae were continuously incubated in 35µm of the wnt antagonist, IWR-1 in 0.25% DMSO. Control larvae were treated with 0.25% DMSO alone. Fresh treatment solutions were added each day after injury until sacrifice and fixation at 8 days post fertilization/3 days post injury. 20 animals were cut to remove a chunk of spinal cord, 1-2 segments caudal and rostral to the injury site, and ventrally, along the notochord. We used the Invitrogen Recover All Total Nucleic Acid Isolation Kit for FFPE to extract the RNA from the tissue samples. This process was repeated for three biological replicates per treatment condition.