Fig. 6

ctgfa⁺ cells are required for SC regeneration. (A) Experimental timeline to evaluate the requirement of ctgfa⁺ cells after SCI. Transgenic ctgfa:mCherry-2A-NTR animals and control siblings were subjected to complete SC transection and metronidazole (MTZ) treatment at 4 and 7 dpi to ablate ctgfa⁺ cells. Swim assays were performed and SC tissues were collected at 14 dpi to assess regeneration. (B-D) Glial bridging in ctgfa:mCherry-2A-NTR animals (Tg⁺) and wild-type siblings (Tg⁻) at 14 dpi. Tg⁺ and Tg⁻ animals were treated with MTZ. Representative immunohistochemistry shows the Gfap⁺ bridge at the lesion site in C. Percentage bridging represents the cross-sectional area of the glial bridge at the lesion site relative to the intact SC 750 µm rostral to the lesion in D. (E-G) Anterograde axon tracing in ctgfa:mCherry-2A-NTR animals (Tg⁺) and wild-type siblings (Tg⁻) at 14 dpi. Biocytin axon tracer was applied rostrally and analyzed at 600 µm (proximal) and 1500 µm (distal) caudal to the lesion (E). Representative images show the extent of biocytin labeling 600 µm (proximal) caudal to the lesion in F. Axon growth was first normalized to the extent of biocytin at 450 and 750 µm rostral to the lesion, and then to labeling in Tg⁻ controls at the proximal level in G. (H) Swim endurance assay for ctgfa:mCherry-2A-NTR animals and wild-type siblings at 14 dpi. This behavioral test was used to assess the capacity of ctgfa-ablated animals to swim in an enclosed swim tunnel under increasing water current velocities. The time at exhaustion for each fish is shown. (I,J) Swim behavior assays were used to assess the performance of ctgfa:mCherry-2A-NTR animals and wild-type siblings at minimal water current velocity at 14 dpi. Average Y-position in the tunnel (I) and burst frequency (J) were quantified at 10 cm/s water current velocity. For all quantifications, dots represent individual animals and animal numbers are indicated in parentheses. Error bars represent s.e.m. ***P<0.001; **P<0.01; *P<0.05 (unpaired t-test with Welch's correction for D and G; one-way ANOVA with multiple comparisons for H-J). Scale bars: 100 µm.