Fig. 2

Contribution of ctgfa⁺ cells to regenerating neurons and oligodendrocytes after SCI. (A) Experimental timeline to evaluate the contribution of ctgfa⁺ cells after SCI. ctgfa-Tracer animals were subjected to complete SC transection and tamoxifen (TAM)-mediated recombination at 4 dpi to enable permanent mCherry labeling in ctgfa⁺ and ctgfa⁺-derived cells. SC tissues were collected at 28 dpi for histological examination. (B) Immunostaining for mCherry (green), HuC/D (magenta) and nuclear Hoechst (gray) at 28 dpi. mCherry expression, referred to as ctgfa-Tracer⁺, is used to trace the fates of ctgfa-expressing cells following TAM-inducible recombination at 4 dpi. SC sections from TAM-treated ctgfa-Tracer (Cre⁺) animals are shown. Cross-sections 150 µm (proximal) or 450 µm (distal) rostral from the lesion site are shown. High-magnification insets show select ctgfa-Tracer⁺ cells in triple-, double- or single-channel views. White arrowheads indicate ctgfa-Tracer⁺ HuC/D⁺ cells; yellow arrowheads indicate ctgfa-Tracer⁻ HuC/D⁺ cells. (C,D) Quantification of ctgfa-Tracer⁺ and HuC/D⁺ colocalization at 28 dpi. ctgfa-Tracer Hoechst and HuC/D Hoechst were used to quantify the numbers of ctgfa-Tracer⁺ and HuC/D⁺ cells. ctgfa-Tracer⁺ HuC/D⁺ cells were normalized to the total number of ctgfa-Tracer⁺ cells in C, and to the total number of HuC/D⁺ cells in D. (E) Immunostaining for mCherry (green), Mbp (magenta) and nuclear Hoechst (gray) at 28 dpi. SC sections from TAM-treated ctgfa-Tracer (Cre⁺) animals are shown. Cross-sections 150 µm (proximal) or 450 µm (distal) rostral from the lesion site are shown. High-magnification insets show select ctgfa-Tracer⁺ cells in triple-, double- or single-channel views. White arrowheads indicate ctgfa-Tracer⁺ Mbp⁺ staining; yellow arrowheads indicate ctgfa-Tracer⁺ Mbp⁻ staining. (F,G) Quantification of ctgfa-Tracer⁺ and Mbp⁺ colocalization at 28 dpi. ctgfa-Tracer⁺ and Mbp⁺ fluorescence were quantified. ctgfa-Tracer⁺ Mbp⁺ cells were normalized to total ctgfa-Tracer⁺ in F and to total Mbp⁺ fluorescence in G. For all quantifications, SC cross-sections 150, 450 and 750 µm rostral to the lesion were analyzed. Dots indicate individual animals and sample sizes are indicated in parentheses. Error bars represent s.e.m. Scale bars: 50 µm.