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Figure 1. Differential regulation of components of the JAK‐STAT pathway in response to SCI in the R and NR stages. (A) Diagram of the JAK‐STAT signaling pathway based on the KEGG database. Components of the pathway are shown in green boxes and, with the exception of receptors, genes with differential expression between the R and NR stages are shown below the boxes. Following the binding of ligands to their respective receptors, STAT proteins are phosphorylated (+P red symbol) and activated by JAK tyrosine kinases. Once activated, STAT proteins dimerize and translocate to the nucleus to modulate gene expression. (B) Heat map of genes related to the JAK‐STAT pathway with differential expression between the R and NR stages. Columns indicate the expression of the R and NR stage at 1, 2, and 6 dpt; grey boxes indicate not detected (ND) transcripts. Orange arrows indicate ligands, blue arrows indicate pathway suppressors and orange T indicate pathway targets. Black lines indicate genes with an early upregulation on R stage (1 and/or 2 dpt) and a sustained or late upregulation on NR stage up to 6 dpt. Dotted lines indicate genes that are only upregulated at 6 dpt on NR stage. According to the Lee‐Liu et al. (2014) data, black and dotted lines consider only statistically significant values and genes identified by Blast2GO are shown in parentheses.
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Figure 2. RT‐qPCR analysis of the levels of JAK‐STAT pathway components in response to SCI. Expression analyses by RT‐qPCR of genes related to the JAK‐STAT pathway are shown in response to SCI for (A) R stage (green), (B) NR56 stage (orange) and (C) NR66 stage (red). At least three biological replicates prepared from different animal pools were analyzed from 6 hpt to 6 dpt or 30 dpt for each stage. Relative expression was normalized to eef1a‐1 expression and to sham operated animals. Two statistical tests were assessed: (i) t‐test with 95% confidence was performed to determine a significant difference (α symbol) from sham levels (log2(transected vs sham) = 0) and (ii) the difference between time points was assessed by multiple comparisons with a one‐way ANOVA test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05).
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Figure 3. Western blot analysis of the activation of the JAK‐STAT pathway in response to SCI. Western blot and histogram analyses showing the levels of phosphorylated STAT3 (pSTAT3) and total STAT3 in the spinal cord after injury and compared with a sham or uncut controls for (A), (B) R stage, (C), (D) NR56 stage and (E), (F) NR66 stage. The presence of STAT3α and STAT3β isoforms is indicated by arrowheads. Western blots are representative of three independent assays for R stage and two assays for each NR stage through 3 hpt and 6 dpt; later times are unique samples. Histograms show the pSTAT3/STAT3β ratio normalized to controls. Control levels (ratio = 1) are indicated by dashed lines. No difference between time points was observed by multiple comparisons with a one‐way ANOVA test.
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Figure 4. Immunofluorescence analysis of p‐STAT3 in R stage animals after SCI. Sections through the spinal cord of regenerative animals analyzed for pSTAT3 (green) and TOTO3 (nuclei, blue). (A), (A′) Sagittal section through the caudal portion of a spinal cord at 3 hpt. White arrows show positions for the stump and 200−600 μm caudal to the lesion site. (B), (B′) Coronal section from an uninjured spinal cord. (C)−(E′) Coronal sections from a transected animal at 3 hpt at (C), (C′) the stump level and (D), (D′) 200 and (E), (E′) 600 μm caudal to the lesion site. Arrows indicate representative pSTAT3+ cells in the ventricular zone (white arrows) and in the grey matter (white arrowheads). (F), (F′) Coronal sections at 1 dpt at 200 μm caudal to the injury site. (G), (G′) Coronal section at 6 dpt at 200 μm caudal to the injury site. Dotted lines delimit the central canal. White bars indicate 100 μm (A), (A′) and 50 μm (B)−(G′). The sections depicted are representative of three animals per time point.
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Figure 5. Immunofluorescence analysis of pSTAT3 levels in NR56 stage animals after SCI. (A) Scheme representing a coronal section of the spinal cord from an NR56 animal: Vz, ventricular zone; Mn, motor neuron columns; Gm, grey matter; Ve, vertebra; Drg, dorsal root ganglion; Nt, notochord. (B)−(E*) Coronal sections through the spinal cord, analyzed for immunostaining of pSTAT3 (green) and TOTO3 staining (nuclei, blue). (B), (B′) Section from a sham operated animal at 1 dpt. (C) (C′) Section from the spinal cord stump at 3 hpt. (D)−(E*) Sections at 3 hpt (D), (D*) and 1 dpt (E), (E*) 200 μm caudal to the lesion; boxed areas indicate a digital zoom of grey matter and motoneuron columns. Arrows indicate representative pSTAT3+ motoneurons (white arrows, cells with large nuclei) and cells in the grey matter (white arrowheads). White bars indicate 100 μm (B)−(D′), (E), (E′) and 30 μm (D′), (D*), (E′), (E*). The sections depicted are representative of two animals per time point.
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Figure 6. Immunofluorescence analysis of pSTAT3 levels in NR66 stage animals after SCI. (A) Scheme representing a coronal section from an NR66 stage spinal cord: Vz, ventricular zone; Mn, motor neuron columns; Gm, remaining grey matter. (B)−(F*) Coronal sections through the spinal cord, analyzed for immunostaining for pSTAT3 (green) and TOTO3 staining (nuclei, blue). (B), (B′) Section from a sham operated animal at 1 dpt. (C), (C*) Section from a transected animal at 3 hpt and 200 μm caudal to the lesion; boxed area indicates a digital zoom of the ventricular zone (vz) and auto‐fluorescent clot (asterisk). (D), (E*) Section at 1 dpt (D), (D*) and 6 dpt (E), (E*) 200 μm caudal to the lesion; boxed areas indicate digital zooms of grey matter and motoneuron columns. Arrows indicate representative pSTAT3+ motoneurons (white arrows, cells with large nuclei) and cells in the grey matter (white arrowheads). (F), (F*) Section at 30 dpt and 100 μm caudal to the lesion; boxed area indicates a digital zoom of the ventricular zone. White bars indicate 100 μm (B), (C′), (D), (D′), (E), (E′), (F), (F′) and 30 μm (C′), (C*), (D′), (D*), (E′), (E*), (F′), (F*). The sections depicted are representative of three animals for 3 hpt to 6 dpt and two animals for 30 hpt.
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Figure 7. JAK‐STAT pathway activation in Sox2/3+ cells. Coronal sections through the spinal cord and injury site of R, NR56 and NR66 animals, analyzed for pSTAT3 (green), Sox2 (red) and Hoechst (nuclei, blue). (A)−(B′) Sections from R stage (A)−(A′) and NR56 stage (B)−(B′) animals at 3 hpt showing the ventricular zone at 300 μm caudal to the lesion site. (C)−(C′) Section from an NR66 stage animal at 15 dpt showing the ventricular zone at 100 μm caudal to the lesion site. The sections depicted are representative of two to three animals. (D), (E) Quantification of ventricular pSTAT3+ Sox2+ cells in one section per animal was performed for (D) R and (E) NR66 stages. Difference between time 012457+\9points was assessed by multiple comparisons with a one‐way ANOVA test (*P < 0.1). (F)−(F’’) Section from an R stage animal at 1 dpt showing the lesion site (ablation gap); representative of three animals. (F), (F′) An optical zoom of (F′). White bars indicate 30 μm.
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Figure 8. Sustained JAK‐STAT pathway activation reduces neurogenesis. (A) Diagram of the spinal cord graft approach is shown. The caudal portion of the thoracic spinal cord was isolated from transgenic tadpoles (identified by GFP+ eyes and represented as red tadpoles) or their control siblings (black tadpoles), and then it was grafted into WT hosts (black). First heat shock was done 30 min before the spinal cord grafting, and then every 2 days. After 5 days the spinal cord grafts were isolated and analyzed by RT‐qPCR. (B) RT‐qPCR analysis for STAT3 targets (socs3, c‐fosa, and c‐myc), NSPC markers (nestin, vima, and vimb), neurogenic genes (ngn2a and ngn3) and astrocyte marker (aldh1l1). Five biological replicates prepared from different animal pools were analyzed. One sample t‐test with 95% confidence was performed to determine a significant difference (* symbol) from control grafts. (C), (D) Representative animals treated as indicated in (A) were analyzed at 14 days post‐grafting. Longitudinal sections of transgenic (C) and control (D) grafts were analyzed by immunofluorescence. Central canal continuity was analyzed by Sox2/Sox3 (red) and axon continuity by nTubulin (green). In (C) transgenic graft (left side of the figure) is connected (indicated by a dotted line) to the caudal portion of the host spinal cord (right side). The section plane of (C) do not include the connection to the rostral side of the host. In (D) control graft is observed in the middle and connected (dotted lines) to the rostral and caudal portions (left and right sides) of the host spinal cord. White bars indicate 30 μm. The sections depicted are representative of three control and transgenic grafts.
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Figure S1. Anatomical positions for spinal cord transection and grafting approaches. A-C, Spinal cord (SC) transection (red scissor and dashed line) is performed at the mid-point between hindlimbs and forelimbs (black dashed lines), as indicated for (A) R-, (B) NR56- and (C) NR66-stages. In control groups (sham operation) an incision in skin and muscle is performed at the same level, without injuring the SC. Caudal portion of the SC is isolated from the transection site to (A) the mid-point of the tail in R-stage, (B) the beginning of the cloaca (Vent tube) in NR56-stage and (C) the end of the spinal cord in NR66-stage animals. D, SC grafting is performed by transections (red scissors and dashed lines) of donor and host in the mid-point between limb buds and in the forelimb buds. SC isolation is performed at the same sites.
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Figure S2. Autofluorescence clots in NR66-stage spinal cord. Coronal sections through
the spinal cord of NR66 animals with focus in the ventricular zone, analyzed for pSTAT3
(green) and Hoescht (nuclei, blue). A-A’, section from an uninjured animal. B-C’, sections
from an animal in the spinal cord stump at 3 hpt. B-B’ shows nuclear signaling of pSTAT3
and C-C’ is the control without primary antibody. White bars indicate 50 μm.
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Figure S3. STAT3 activation in motoneurons and sensory neurons. Coronal sections
through the spinal cord of R- and NR56-stage animals, analyzed for pSTAT3 (green),
Islet1/2 (red) and Hoescht (nuclei, blue). Motoneuron columns are indicated by mn and
dorsal root ganglia by drg. A-B’’, sections from R-stage animals at 3hpt (A-A’’) and 1dpt
(B-B’’). C-F’’, sections from NR56-stage animals at 3hpt (C-C’’) and 1dpt (D-D’’). White
bars indicate 50 μm (A-B’’) and 100 μm (C-D’’).
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Figure S4. STAT3 activation in leukocytes. Coronal sections through the spinal cord of
R- and NR66-stage animals, analyzed for pSTAT3 (green), CD45 (red) and Hoescht
(nuclei, blue). Leukocytes pSTAT3+ are indicated by white asterisks in the meninges and
by white arrows in the spinal cord. A-B’, sections from an uninjured R animal (A) and at
1dpt (B-B’). C-E’, sections from an uninjured NR66 animal (C) and at 1dpt (E-E’). White
bars indicate 50 μm in A-Bᶧ, 100 μm in C-D and 30 μm in E-F. C-F, quantification of
pSTAT3+ CD45+ cells in three animals per time point was performed for (C) R- and (F)
NR66-stages. Difference between time points was assessed by multiple comparisons with a
one-way ANOVA test (P value **>0,01 and *<0,1).
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Figure S5. Characterization of Tg(HS:caSTAT3GR) transgenic tadpoles. A. Transgenic
construct used in this study. caSTAT3GR was inserted in an HGEM construct by HindIII
and BssHII restriction enzymes. B-E, F1 transgenic animals and their control siblings were
raised to stage 50, heat shocked once and incubated with dexamethasone during 2 days. B,
RT-qPCR analysis shows gr up-regulation in the central nervous system (CNS) 6 hours and
1 day post-treatment C, Western blot analysis shows presence of caSTAT3GR protein (red
arrow, GR) in comparison to total STAT3 (black arrows, α and β isoforms) at 8 hours posttreatment
in the CNS and in the tail. D, RT-qPCR analyses shows socs3 up-regulation in
the CNS after treatment. E, Tadpoles expressing caSTAT3GR had a reduced survival rate
in comparison with their controls siblings. For RT-qPCR analyses, one sample t-test with
95% confidence was performed to determine a significant difference (* symbol) from
control tadpoles, using at least 3 independent replicates.
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