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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 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. |
