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. 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. 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. 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.

中文翻译：

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非洲爪蟾再生和非再生阶段脊髓损伤的细胞反应

幼虫阶段的有效再生能力以及小蛙变态后的非再生反应使非洲爪蟾成为了解导致脊髓再生的细胞反应的理想模型生物。我们比较了非洲爪蟾再生和非再生阶段对脊髓损伤的细胞反应。对于该分析，我们使用了细胞外基质的电子显微镜、免疫荧光和组织学染色。我们生成了两个转基因系：i) 带有驱动 EGFP 表达的斑马鱼 GFAP 调节区的报告系，以及 ii) 具有相同 GFAP 调节区的细胞特异性诱导消融系。此外，我们使用 FACS 分离 EGFP+ 细胞进行 RNAseq 分析。在再生阶段的动物中，脊髓再生触发了受伤残端的快速封闭，随后中央管内的细胞增殖，并在消融间隙中形成玫瑰花状结构。此外，中央管中充满了与中央管、神经元、轴突甚至突触结构的细胞形态相似的细胞。受伤后 20 天后几乎完成再生。在非再生阶段的动物中，观察到大部分受损的组织，没有明显的残端闭合。消融间隙充满成纤维细胞样细胞和细胞外基质成分的沉积。即使在受伤后 40 天后也没有观察到脊髓的重建。细胞标志物分析证实了这些组织学差异，波形蛋白的短暂增加，在再生阶段检测到纤连蛋白和胶原蛋白，这与大多数这些标志物的持续积累相反，包括在 NR 阶段的硫酸软骨素蛋白聚糖。斑马鱼 GFAP 转基因系已经过验证，我们已经证明这是研究神经干祖细胞 (NSPC) 作用的一种非常可靠的新工具。GFAP::EGFP 细胞的 RNASeq 使我们能够清楚地证明这些细胞确实是 NSPC。相反，GFAP::EGFP转基因主要在非再生阶段的星形胶质细胞中表达。在再生阶段，脊髓损伤会激活 NSPCs 的增殖，我们发现它们主要分化为神经元和神经胶质细胞。这些细胞的特异性消融消除了适当的再生，证实 NSPCs 细胞是脊髓功能再生所必需的。在再生和非再生阶段，细胞对脊髓损伤的反应在两个阶段之间有很大的不同。再生反应的一个关键标志是 NSPCs 的激活，它大量增殖并分化为神经元以重建脊髓。同样值得注意的是，在再生阶段没有观察到胶质瘢痕形成，但在非再生阶段的动物中会形成短暂的胶质瘢痕状结构。并分化成神经元以重建脊髓。同样值得注意的是，在再生阶段没有观察到胶质瘢痕形成，但在非再生阶段的动物中会形成短暂的胶质瘢痕状结构。并分化成神经元以重建脊髓。同样值得注意的是，在再生阶段没有观察到胶质瘢痕形成，但在非再生阶段的动物中会形成短暂的胶质瘢痕状结构。