Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

Abstract

Background The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration. Methods We compared the cellular response to spinal cord injury between the regenerative and non-regenerative stages of Xenopus laevis. For this analysis, we used electron microscopy, immunofluorescence and histological staining of the extracellular matrix. We generated two transgenic lines: i) the reporter line with the zebrafish GFAP regulatory regions driving the expression of EGFP, and ii) a cell specific inducible ablation line with the same GFAP regulatory regions. In addition, we used FACS to isolate EGFP+ cells for RNAseq analysis. Results In regenerative stage animals, spinal cord regeneration triggers a rapid sealing of the injured stumps, followed by proliferation of cells lining the central canal, and formation of rosette-like structures in the ablation gap. In addition, the central canal is filled by cells with similar morphology to the cells lining the central canal, neurons, axons, and even synaptic structures. Regeneration is almost completed after 20 days post injury. In non-regenerative stage animals, mostly damaged tissue was observed, without clear closure of the stumps. The ablation gap was filled with fibroblast-like cells, and deposition of extracellular matrix components. No reconstruction of the spinal cord was observed even after 40 days post injury. Cellular markers analysis confirmed these histological differences, a transient increase of vimentin, fibronectin and collagen was detected in regenerative stages, contrary to a sustained accumulation of most of these markers, including chondroitin sulfate proteoglycans in the NR-stage. The zebrafish GFAP transgenic line was validated, and we have demonstrated that is a very reliable and new tool to study the role of neural stem progenitor cells (NSPCs). RNASeq of GFAP::EGFP cells has allowed us to clearly demonstrate that indeed these cells are NSPCs. On the contrary, the GFAP::EGFP transgene is mainly expressed in astrocytes in non-regenerative stages. During regenerative stages, spinal cord injury activates proliferation of NSPCs, and we found that are mainly differentiated into neurons and glial cells. Specific ablation of these cells abolished proper regeneration, confirming that NSPCs cells are necessary for functional regeneration of the spinal cord. Conclusions The cellular response to spinal cord injury in regenerative and non-regenerative stages is profoundly different between both stages. A key hallmark of the regenerative response is the activation of NSPCs, which massively proliferate, and are differentiated into neurons to reconstruct the spinal cord. Also very notably, no glial scar formation is observed in regenerative stages, but a transient, glial scar-like structure is formed in non-regenerative stage animals.

Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate Central Nervous System

“…, nucleobase-containing small molecule metabolic process, DNA repair, DNA conformation change, AURORA PATHWAY, base excision repair). Such categories were consistent with the increased proliferation of macrophages and other myeloid cell types, reactive stem cells, and the enhanced epigenetic reprogramming that occurs in regenerative nervous systems [ 5 , 27 , 77 , 117 , 127 ]. Because of the fewer number of genes involved, hypermethylated genes exhibiting decreased RNA expression were enriched with generally lower probability (4 < -log 10 (P) < 9) for categories associated with physiological functions ( e.g.…”

“…These physiological processes are activated in axotomized neurons and their surrounding support cells. For example, successful CNS regeneration in Xenopus is supported by activation of proliferative macrophages and radial glia [ 27 , 77 , 127 ], and many genes activated in cancer metastasis are also involved in axon outgrowth ( e.g. , [ 7 ]).…”

New insights into glial scar formation after spinal cord injury

“…The ability to regenerate in fish is, in part, due to their capacity for scar-free wound healing (Tsata et al 2020). The loss of regeneration in amphibians occurs at stages when they begin developing scar-like tissue, likely due to maturation of the immune system (Bertolotti et al 2013, Edwards-Faret et al 2021.…”

Dynamic Diversity of Glial Response Among Species in Spinal Cord Injury

“…Comparison between regenerative (pre-metamorphosis stages) and non-regenerative (during metamorphosis) responses in Xenopus laevis highlighted that, in the same species, no glial scar was observed in regenerative stages conversely to the nonregenerative stage where a transient glial scar-like structure was formed (Edwards-Faret et al, 2021). Similarly, spinal crush injury in neonatal mice up to postnatal Day 2 led to scar-free healing, allowing axonal regrowth through the lesion (Li et al, 2020).…”