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Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate (Xenopus laevis) Central Nervous System Characterized by Whole Genome Bisulfite Sequencing (WGBS)

ABSTRACT: Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate (Xenopus laevis) Central Nervous System Characterized by Whole Genome Bisulfite Sequencing (WGBS)

PROVIDER: PRJNA760352 | ENA |

REPOSITORIES: ENA

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Project description:BackgroundBecause some of its CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.g., hindbrain neurons after spinal cord injury (SCI)) lose this capacity as tadpoles metamorphose into frogs, the South African claw-toed frog, Xenopus laevis, offers unique opportunities for exploring differences between regenerative and non-regenerative responses to CNS injury within the same organism. An earlier, three-way RNA-seq study (frog ONC eye, tadpole SCI hindbrain, frog SCI hindbrain) identified genes that regulate chromatin accessibility among those that were differentially expressed in regenerative vs non-regenerative CNS [11]. The current study used whole genome bisulfite sequencing (WGBS) of DNA collected from these same animals at the peak period of axon regeneration to study the extent to which DNA methylation could potentially underlie differences in chromatin accessibility between regenerative and non-regenerative CNS.ResultsConsistent with the hypothesis that DNA of regenerative CNS is more accessible than that of non-regenerative CNS, DNA from both the regenerative tadpole hindbrain and frog eye was less methylated than that of the non-regenerative frog hindbrain. Also, consistent with observations of CNS injury in mammals, DNA methylation in non-regenerative frog hindbrain decreased after SCI. However, contrary to expectations that the level of DNA methylation would decrease even further with axotomy in regenerative CNS, DNA methylation in these regions instead increased with injury. Injury-induced differences in CpG methylation in regenerative CNS became especially enriched in gene promoter regions, whereas non-CpG methylation differences were more evenly distributed across promoter regions, intergenic, and intragenic regions. In non-regenerative CNS, tissue-related (i.e., regenerative vs. non-regenerative CNS) and injury-induced decreases in promoter region CpG methylation were significantly correlated with increased RNA expression, but the injury-induced, increased CpG methylation seen in regenerative CNS across promoter regions was not, suggesting it was associated with increased rather than decreased chromatin accessibility. This hypothesis received support from observations that in regenerative CNS, many genes exhibiting increased, injury-induced, promoter-associated CpG-methylation also exhibited increased RNA expression and association with histone markers for active promoters and enhancers. DNA immunoprecipitation for 5hmC in optic nerve regeneration found that the promoter-associated increases seen in CpG methylation were distinct from those exhibiting changes in 5hmC.ConclusionsAlthough seemingly paradoxical, the increased injury-associated DNA methylation seen in regenerative CNS has many parallels in stem cells and cancer. Thus, these axotomy-induced changes in DNA methylation in regenerative CNS provide evidence for a novel epigenetic state favoring successful over unsuccessful CNS axon regeneration. The datasets described in this study should help lay the foundations for future studies of the molecular and cellular mechanisms involved. The insights gained should, in turn, help point the way to novel therapeutic approaches for treating CNS injury in mammals.

Project description: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.

Dataset Information

Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate (Xenopus laevis) Central Nervous System Characterized by Whole Genome Bisulfite Sequencing (WGBS)

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