April 26, 2012;
Spinal cord regeneration in Xenopus tadpoles proceeds through activation of Sox2-positive cells.
In contrast to mammals, amphibians, such as adult urodeles (for example, newts) and anuran larvae (for example, Xenopus) can regenerate their spinal cord
after injury. However, the cellular and molecular mechanisms involved in this process are still poorly understood. Here, we report that tail
amputation results in a global increase of Sox2
levels and proliferation of Sox2
(+) cells. Overexpression of a dominant negative form of Sox2
diminished proliferation of spinal cord
resident cells affecting tail
regeneration after amputation, suggesting that spinal cord
regeneration is crucial for the whole process. After spinal cord
(+) cells are found in the ablation gap forming aggregates. Furthermore, Sox2
levels correlated with regenerative capabilities during metamorphosis, observing a decrease in Sox2
levels at non-regenerative stages. Sox2
(+) cells contribute to the regeneration of spinal cord
amputation and transection. Sox2
levels decreases during metamorphosis concomitantly with the lost of regenerative capabilities. Our results lead to a working hypothesis in which spinal cord
damage activates proliferation and/or migration of Sox2
(+) cells, thus allowing regeneration of the spinal cord
amputation or reconstitution of the ependymal epithelium
after spinal cord
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References [+] :
Figure 1. Sox2 is upregulated after tail amputation. (A,B) Immunofluorescence of transverse sections of stage 49 tadpoles with anti-Sox2 (red) and anti-acetylated α-tubulin (green) antibodies; a 20 μm Z-projection was made to detect cilia (arrow). DNA was counterstained with TOTO3 (blue). (B) Magnification of the inset indicated in (A). (C) Reverse transcription polymerase chain reaction (RT-PCR) analysis of sox2 and Xenopus brachyury (Xbra) mRNA levels at the tip of the regenerating tail indicating that sox2 is upregulated after tail amputation. EF1α was used as loading control. (D-F)In situ hybridization using an antisense probe against sox2 during tail regeneration of stage 49 tadpoles at (D) 3, and (E,F) 7 days post amputation (dpa), sox2 was detected on the spinal cord and (D, inset) neural ampulla. (F) Transversal section of the regenerating tail at 7 dpa at the level indicated in (E), arrow indicates sox2 staining in the ependymal epithelium. (G-L) Sagittal optical section of whole-mount immunofluorescence with anti-Sox2 antibodies (red) before amputation (G,H) and at 4 dpa (I,J) rostral and (K,L) caudal regions. DNA was stained in blue. (H,J,L) Pseudocolored intensities of Sox2 channel from (G), (I) and (K), respectively as is described in the left scheme. Arrowheads: amputation plane. Scale bars: (A) 25 μm, (D,E) 400 μm, (F-L) 50 μm.
Figure 2. Systemic upregulation of Sox2 after tail amputation. (A,B) Tails from stage 49 tadpoles at 3 days post amputation (dpa). Dorsal (top) and lateral (bottom) view. (A) sox2 in situ hybridization and (B) 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI) vital dye staining to identify the lateral line. Arrowheads indicate the amputation plane. (C-F) Dorsal view of sox2 whole mount in situ hybridization of the cephalic region of (C) non-amputated tadpoles, and tadpoles at (D) 3, (E) 7 and (F) 21 dpa. Insets show higher magnification of the lateral line and olfactory epithelial region that increase sox2 expression at 3 and 7 dpa. Notice that after fixation, tails were removed from their base and the observed stump ends do not correspond to the original amputation. (G) Diagram depicting the experimental set up for (H-K). (H-K)sox2 in situ hybridization in areas 5 to 8 mm rostral to the amputation plane from tadpoles at different dpa. sox2 is present in the spinal cord at 3 and 7 dpa (indicated by arrowheads). Endogenous expression of alkaline phosphatase in the notochord has been described previously  suggesting that expression on this tissue is non-specific. (L) Diagram depicting the experimental set up for (M). (M) Reverse transcription polymerase chain reaction (RT-PCR) analysis of sox2 mRNA levels in isolated spinal cord indicates that sox2 is upregulated in a rostral region during tail regeneration. EF1α was used as loading control. Scale bars: (A,B) 400 μm, (C-F) 400 μm, (H-K) 100 μm.
Figure 3. Tail amputation increases proliferation of Sox2+cells. (A) Diagram showing the experimental set up to evaluate incorporation of 5-bromo-2'-deoxyuridine (BrdU) after tail amputation of stage 49 tadpoles. Each box represents 1 day, red boxes indicate BrdU incubation (pulse) and blue circles the day of tail fixation (chase). Tadpoles were incubated in BrdU for 24 h chased at different days post amputation (dpa). Then they were processed for coimmunostaining against Sox2 and BrdU and analyzed as described in (C-P). (B) Diagram of the rostral, cut and caudal regions analyzed in (C-O). (C-N) Sagittal optical section of whole-mount immunofluorescence against BrdU (green) and Sox2 (red) double immunofluorescence from tails labeled as depicted in (A) and fixed at (C-E) 0, (F-H) 2, (I-K) 4 and (L-N) 7 dpa in the cut region (C-K) and caudal region (L-N). DNA is stained in blue. During regeneration, an increase of Sox2+/BrdU+ cells was observed in the spinal cord and (I-N) neural ampulla. Arrowheads indicate the amputation plane. (O) Graph describing the percentage of Sox2+/BrdU+ cells in the overall Sox2+ cells at proximal, cut and caudal regions of spinal cord at different dpa. An increase Sox2+ cell proliferation was observed in all the regions mostly at 4 to 7 dpa. (P) Graph showing the percentage of BrdU+ cells in the spinal cord, notochord and mesenchymal cells at different days post amputation quantified at the cut level. (O,P) The number of tadpoles counted for each point (n) is indicated above the bars. Scale bar: 25 μm.
Figure 4. Overexpression of a dominant negative form of Sox2 impairs tail regeneration. (A-F) Sagittal optical sections showing the predominant phenotype of regenerated tails in (A-C) enhanced green fluorescent protein (EGFP; total regeneration) and (D-F), dnSox2; EGFP (no regeneration) F0 transgenic tadpoles amputated at stage 42. (A,B,D,E) Images showing transgenic EGFP expression (green) and DNA (red) staining. (B,E) Higher magnifications from the areas indicated in (A) and (D), respectively. (C,F) Differential interference contrast (DIC) microscopy from (A) and (D), respectively. (G) Graph showing the regenerative efficiency of EGFP and dnSox2; EGFP stage 42 transgenic tadpoles. White bars represent the percentage of regeneration and black bars the score of regeneration. dnSox2; EGFP tadpoles have a decrease of regenerative efficiency compared to EGFP. (H-O) 5-Bromo-2'-deoxyuridine (BrdU) incorporation in (H-K) EGFP and (L-O) dnSox2;EGFP F0 transgenic stage 49 tadpoles at 4 days post amputation (dpa) and (J,N) DIC microscopy from tails depicted in (H) and (L). (K-O) Magnification of the zone labeled in (I) and (M). (P) Graph showing the percentage of BrdU+ cells in the spinal cord from high EGFP expressing transgenic tadpoles represented in (K) and (O). BrdU incorporation was diminished in dnSox2; EGFP compared to EGFP controls. (Q) Regenerative efficiency of EGFP and dnSox2; EGFP transgenic tadpoles amputated at stage 48, a decrease in the score of regeneration was observed in dnSox2; EGFP. (R) Graph showing that the regenerated spinal cord length of dnSox2; EGFP transgenic tadpoles decreased compared to controls (EGFP). The length of the spinal cord was corrected by the tail width. (G,P-R) The number of tadpoles analyzed (n) is indicated above the bars. Scale bars: 50 μm.
Figure 5. Spinal cord regeneration and Sox2 expression during metamorphosis. (A-D) Immunofluorescences of stage 50 tadpoles against acetylated α-tubulin (green) on horizontal sections from the injured area at (A) 0 days post transection (dpt) with paraplegia phenotype, (B) 0 days post sham operation (dps) that has coordinated swimming, (C) 7 dpt with circular swimming and (D) 28 dpt with coordinated swimming. Insets in (B-D) show 1.5 times magnifications of the areas contained in the frames, which correspond to the transection levels. (E-H) Graphs showing functional recovery of tadpoles transected at different metamorphic stages (st). Functional recovery decreases meanwhile metamorphosis undergoes. (I) Diagram depicting the isolation of spinal cord samples used for J,K. (J,K) Western blot analysis of neural-related markers (J) Sox2, glial fibrillary acidic protein (GFAP), (K) Nestin and Vimentin at different stages of metamorphosis. Scale bars: 50 μm.
Figure 6. Response of Sox2+cells to spinal cord transection. (A-L) Tadpoles were incubated with 5-bromo-2'-deoxyuridine (BrdU) between days 2 and 4 after (A-D) sham operation or (E-L) spinal cord transection. Horizontal sections from animals fixed at 4 days post sham operation (dps) or days post transection (dpt) were immunostained for BrdU (green), Sox2 (red) and DNA (blue). (E-H) Transection zone and (I-L) region rostral to transection zone. (M,N) Magnification of (J) and (L). White arrows indicate Sox2+/BrdU- cells distant to ependyma, and Sox2+/BrdU+ cells are indicated by yellow arrows. (O) Sox2 immunofluorescence on a horizontal section from tadpoles at 11 dpt. Inset: magnification of the ablation gap that contains Sox2+ cells aggregates (arrows). (P) Analysis of the percentage of Sox2+/BrdU+ over total number of Sox2+ cells from samples represented in A-L. Sox2+ cell proliferation increase at the rostral level of the transection site. Scale bars: (D,H,L) 50 μm, (N,O) 25 μm.
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