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| Status |
Public on Jan 05, 2021 |
| Title |
Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis. |
| Organism |
Xenopus laevis |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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 fated to form 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 to reconstitute the spinal cord, and are differentiated into neurons. 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.
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| Overall design |
Characterization of cell population GFAP::EGFP from the transgenic line Xla.Tg(Dre.gfap:EGFP)Larra in regenerative stages of Xenopus laevis
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| Contributor(s) |
Edwards-Faret G, González-Pinto K, Cebrián-Silla A, Peñailillo J, García-Verdugo JM, Larraín J |
| Citation(s) |
33526076 |
| Submission date |
Jan 04, 2021 |
| Last update date |
Apr 06, 2021 |
| Contact name |
Juan Larraín |
| E-mail(s) |
jlarrain@bio.puc.cl
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| Organization name |
Pontificia Universidad Catolica de Chile
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| Department |
Cellular and Molecular Biology
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| Lab |
Developmental and Regenerative Biology
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| Street address |
Portugal 49
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| City |
Santiago |
| ZIP/Postal code |
8330025 |
| Country |
Chile |
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| Platforms (1) |
| GPL22393 |
Illumina HiSeq 4000 (Xenopus laevis) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA689548 |
| SRA |
SRP300206 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE164204_RAW.tar |
690.0 Kb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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