Additional file 8 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
Additional file 8: Supplementary Table 1. List of genes, ID number and their respective primer-Forward and primer-Reverse used for RT-qPCR analysis.
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…
Cellular composition and organization of the spinal cord central canal during metamorphosis of the frogXenopus laevis
Studying the cellular composition and morphological changes of cells lining the central canal during Xenopus laevis metamorphosis could contribute to understand postnatal development and spinal cord regeneration. Here we report the analysis of central canal cells at different stages during metamorphosis using immunofluorescence for protein markers expression, transmission and scanning electron microscopy and cell proliferation assays. The central canal was regionalized according to expression of glial markers, ultrastructure, and proliferation in dorsal, lateral, and ventral domains with differences between larvae and froglets. In regenerative larvae, all cell types were uniciliated, have a…
Additional file 6 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
Additional file 6: Figure S6. Analysis of EdU+ cells in the intestine. (A-B) Click-iT staining of EdU+ (red) of the intestine in (A) sham control animals (2 dps), and at (B) 2 dpt. Nuclei were stained with Hoechst (blue). (C) Graph of EdU+ cells per mm3 in the intestine. n = 3.
Additional file 4 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
Additional file 4: Figure S4. Transgenic line Xla.Tg(Dre.gfap:EGFP)Larra. (A-C) Three different animals’ electroporated in the spinal cord with the CAG promoter driving the expression of EGFP in central canal cells. (D-F) Three different animals electroporated in the spinal cord with the zGFAP::EGFP construct driving specific expression in radial glial like cells in contact with the central canal. (G-J) Animals at different developmental stages of the transgenic line Xla.Tg(Dre.gfap:EGFP)Larra showing expression of EGFP in the neural tube at (G-G’) NF stage 23; (H-H′) NF stage 27; (I-I′) NF stage 31 and in the CNS at (J-J’) NF stage 41. (K-M) Double staining against (K) EGFP and (L) Sox2 in…
Additional file 7 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
Additional file 7: Figure S7. Transgenic line Xla.Tg(Dre.gfap:mCherry-Nitroreductase) allows selective cell ablation. (A) Diagram of injection and electroporation of the spinal cord at NF stage 50, indicating volume, concentration and parameters of electroporation. (B) Scheme of electroporation of the Dre.gfap:mCherry-Nitroreductase construct and treatment with vehicle or metronidazol (MTZ) at NF stage 50. (C-R) mCherry (red) expression in the spinal cord of animal electroporated at (C-D; I-J) 2 days post electroporation (dpe), before treatment; (E-F; K-L) 4 dpe and 2 days post treatment (dtt); (G-H; M-N) 7 dpe and 5 dtt, and (O- R) at 8 dpe and 6 dtt co-stained with Hoechst (blue). (S) The…
Additional file 5 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
Additional file 5: Figure S5. RNAseq of EGFP+ and EGFP− cells isolated from the transgenic line Xla.Tg(Dre.gfap:EGFP)Larra. (A) Flow chart of RNAseq bioinformatics analysis from EGFP+ and EGFP− cells. (B) Graph of the Log2 fold change of the differential gene expression between EGFP+ cells versus EGFP− cells after FACS and RNAseq. EGFP expression in EGFP+ cells (green) is highlighted.
Additional file 2 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
Additional file 2: Figure S2. In vivo time-lapse imaging of cells being extruded into the central canal. (A) Rostral stump of the transected spinal cord from a zGFAP::EGFP transgenic animal at R-stage 2 dpt. A time-lapses during 7 h for EGFP and transmitted light (T-PMT) z-stack were capture at the following time points: (B-B′) 0 min; 60 min (C-C′); 120 min (D-D′); 180 min (E-E’); 240 min (F-F′); 300 min (G-G’); 360 min (H-H′). White and purple arrows point to extrusion events from the cells lining the central canal.
Additional file 1 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
Additional file 1: Figure S1. Cellular response to spinal cord injury in R- and NR-stages. (A) Centriolar satellite ultrastructure (arrowheads) in cells surrounding the rostral stump. (B) Radial projection of cells lining the central canal (yellow shadow). (C) Neutrophil in the injury site at 2 dpt in animals at NF stage 50. (C-E) Cells lining a rosette structure at 6 dpt are characterized by a (D) basal collagen lamina (blue shadow), (E) interdigitations and adherent junctions (arrowheads), and (F) intermediate filaments (arrowheads). Graphs of the number of red blood cells/μm2 × 105 at (G) 2 and 6 dpt in NF stage 50, and (H) at 2 and 6 dpt in NF stage 66. Graphs of the number of macrophag…
Additional file 3 of Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
Additional file 3: Figure S3. Quantification of Vimentin Western Blot and Collagen AFOG staining. Western blot replicates for Vimentin and GAPDH in uninjured animals (ui), and after 2, 6, 10, 20 dpt in (A, B) R-Stage and (C, D) NR-Stage. Graphs of the adjusted relative density bands of Vimentin to the GAPDH control and normalized to the uninjured sample (ui) in (E) R-stage and (F) NR-stage at 2, 6, 10 and 20 days post transection (dpt) spinal cord samples. (G) Graph of the adjusted collagen staining area relative to the uninjured (ui) animals at 6, 10 dpt of R-stage and 10, 20 dpt of NR-stage. Red line defined no changes of Vimentin levels or Collagen staining. t-Test: * p