Genome-wide expression profile of the response to spinal cord injury in Xenopus laevis reveals extensive differences between regenerative and non-regenerative stages

Abstract

Background: Xenopus laevis has regenerative and non-regenerative stages. As a tadpole, it is fully capable of functional recovery after a spinal cord injury, while its juvenile form (froglet) loses this capability during metamorphosis. We envision that comparative studies between regenerative and non-regenerative stages in Xenopus could aid in understanding why spinal cord regeneration fails in human beings.

Results: To identify the mechanisms that allow the tadpole to regenerate and inhibit regeneration in the froglet, we obtained a transcriptome-wide profile of the response to spinal cord injury in Xenopus regenerative and non-regenerative stages. We found extensive transcriptome changes in regenerative tadpoles at 1 day after injury, while this was only observed by 6 days after injury in non-regenerative froglets. In addition, when comparing both stages, we found that they deployed a very different repertoire of transcripts, with more than 80% of them regulated in only one stage, including previously unannotated transcripts. This was supported by gene ontology enrichment analysis and validated by RT-qPCR, which showed that transcripts involved in metabolism, response to stress, cell cycle, development, immune response and inflammation, neurogenesis, and axonal regeneration were regulated differentially between regenerative and non-regenerative stages.

Conclusions: We identified differences in the timing of the transcriptional response and in the inventory of regulated transcripts and biological processes activated in response to spinal cord injury when comparing regenerative and non-regenerative stages. These genes and biological processes provide an entry point to understand why regeneration fails in mammals. Furthermore, our results introduce Xenopus laevis as a genetic model organism to study spinal cord regeneration.

Figure 1

Spinal cord transection and regeneration in Xenopus laevis regenerative and non-regenerative stages. (a,b) Xenopus laevis R-stage (a) and NR-stage (b), indicating the transection site at the mid-thoracic point (green and red arrows). (c-j) Immunofluorescence in longitudinal spinal cord sections using an anti-acetylated tubulin antibody to stain for axon growth after the injury. Insets show magnifications of boxed areas. While axon growth occurs across the lesion site in the R-stage, this is not the case for the NR-stage. Brackets, ablation gap. Arrowheads, wrapping of axons around the stump. Arrow, wisping or axonal growth tips into the ablation gap. Scale bars: (a, b) 1 cm, (c-j) 100 μm. dpt, days post transection; NR, non-regenerative; R, regenerative.

Figure 2

Differentially expressed transcripts in response to spinal cord transection. (a) Samples were isolated after 1, 2, or 6 days post-surgery (transection or sham) from regenerative and non-regenerative animals. (b) MA plots depicting differentially expressed transcripts. Blue dots and numbers correspond to transcripts upregulated after transection and orange dots and numbers to those downregulated after transection.

Figure 3

Comparison of the response to spinal cord injury in regenerative and non-regenerative stages. (a) Venn diagram showing all differentially expressed transcripts detected in all time points in R- (green) and NR-stage animals (red). Out of a total of 7,431 transcripts detected in all samples as differentially expressed, 18.9% were differentially regulated in both stages, 29.6% regulated exclusively in the R-stage and 51.5% in the NR-stage. (b, c) Total transcripts that responded differently in R- (b) and NR-stages (c) are depicted as bar graphs. Blue bars, upregulated transcripts; yellow bars, downregulated transcripts.

Figure 4

Differential regulation of genes related to neurogenesis and the axonal growth cone. (a) Heat map and clustering analysis of differential expression of genes related to neurogenesis, showing one cluster of genes exclusively upregulated in the R-stage (I) and another that shows an early upregulation or no change in the R-stage and a delayed upregulation in the NR-stage (II). (b) Heat map and clustering analysis of differentially expressed genes expressed in the axonal growth cone, showing downregulation of a large cluster of genes in the NR-stage 6 days after injury (III). GenBank IDs are shown in square brackets.

Figure 5

Gene ontology enrichment analysis for upregulated transcripts. Gene ontology (GO) enrichment analysis was performed for transcripts that showed a different response to injury when comparing regenerative and non-regenerative stages for Day 1 (a, b), Day 2 (c, d), and Day 6 (e, f). Colors classify GO terms into the following categories: cell cycle, blue; response to stress, pink; developmental processes, orange; metabolic processes, purple; immune response and inflammation, green; others, grey.

Figure 6

Gene ontology enrichment analysis for downregulated transcripts. Gene ontology (GO) enrichment analysis was performed for transcripts that showed a different response to injury when comparing regenerative and non-regenerative stages for Day 1 (a, b), Day 2 (c, d), and Day 6 (e, f). Colors classify GO terms into the following categories: cell cycle, blue; response to stress, pink; developmental processes, orange; metabolic processes, purple; immune response and inflammation, green; others, grey.

Figure 7

RT-qPCR data validation for transcripts from gene ontology categories. Bars show RT-qPCR results for at least two biological replicates prepared from different pools of animals, validating differential expression changes for transcripts from biological processes. (a) Metabolic processes. (b) Immune response and inflammation. (c) Cell cycle. (d) Response to stress. (e) Developmental processes. (f) Hypothetical proteins. Green line, RNA-Seq results for the regenerative stage; red line, RNA-Seq results for the non-regenerative stage. GenBank IDs are shown in square brackets.