Fukuoka T, Kato A, Hirano M, Ohka F, Aoki K, Awaya T, Adilijiang A, Sachi M, Tanahashi K, Yamaguchi J, Motomura K, Shimizu H, Nagashima Y, Ando R, Wakabayashi T, Lee-Liu D, Larrain J, Nishimura Y, Natsume A.

	Figure 1. Comparison of the candidate gene expressions in the acute phase between Xenopus laevis tadpoles and aged mice after spinal cord injury(A) The nonclustering heatmap shows the differential expression of basic-helix-loop-helix (bHLH) transcription factors during the regenerative (R) and nonregenerative (NR) stages in the spinal cord after injury in Xenopus laevis (X. laevis).(B) Quantification of mRNA expression levels for candidate genes (Neurod4, Neurod1, Atoh1, Neurog2, Ascl1) in the mouse SCI model. Gene expression levels were compared to those of the Sham (n = 3 mice per group). Statistical analysis was performed using student's t-test: ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001 and N.S. = not significant. P = 0.1770 to 1 DPI, 0.7074 to 3 DPI in Neurod4; P = 0.0225 to 1 DPI, 0.2071 to 3 DPI in Neurod1; P = 0.0022 to 1 DPI, 0.0328 to 3 DPI in Atoh1; P = 0.0073 to 1 DPI, 0.0329 to 3 DPI in Neurog2; P < 0.0001 to 1 DPI, 0.1935 to 3 DPI in Ascl1. Data represent the mean ± S.D.(C) Superior expression of genes of X. laevis tadpole (in both L and S chromosomes) to aged mice in the stage for nerve regeneration. The bars indicate that the sum of FPKM ratios in the injured group over the sham group in X. laevis after SCI at 1 and 2 DPIs in the R stage (R1 +R2) was divided by the sum of qPCR values in the SCI group over the sham group at 1 and 3 DPIs (1 DPI +3 DPI). FPKM, fragments per kilobase of transcript per million. DPI, days post injury.See also Table S1.
	Figure 2. Preferential transduction of pseudotyped retrovirus with envelope of Lymphocytic choriomeningitis virus tropic to neural stem cells(A) Representative images of AcGFP1 (an aqueous green fluorescent protein) (green), BrdU staining (red), and DAPI (blue) of cells around the ependymal cells lining the central canal (CC) in the sham and the SCI at 3 and 5 DPI. High-magnification images of the center panels are shown in the right panels. Arrowheads indicate cells that are both virus infected (AcGFP1-positive) and dividing (BrdU-positive), these can be observed around the CC of SCI at 3 and 5 DPI; however these cannot be observed in the sham.(B) Representative images of tauAcGFP1 (green), Nestin (red), and DAPI (blue) in the sham and the SCI at 7 DPI. A high-magnification image of the center panel is shown in the right panel. Arrowheads indicate cells that are virus infected (tauAcGFP1-positive) and Nestin-positive neural stem cells. The graphs show the percentage of Nestin-positive cells around the CC in the sham and the SCI groups at 7 DPI. The number of Nestin-positive cells in four separated fields were counted in the sham and the SCI groups at 7 DPI (Figure S2C). The efficiency of the virus in infecting activated neural stem cells is calculated as the ratio of green fluorescent protein (GFP)-positive cells to Nestin-positive cells. Statistical analysis was performed using student's-t test: ∗∗∗∗p < 0.00005. Data represent the mean ± S.D. CC, central canal; BrdU, Bromodeoxyuridine, 5-bromo-2′-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole.See also Figures S2 and S3.
	Figure 3. Differentiation of activated neural stem cells into NeuN- and DCX-positive neurons by introducing Neurod4 into mice(A) Neuronal differentiation from Neurod4-introduced neural stem cells at 5, 7, and 42 DPI. Representative images of AcGFP1 (green) and DCX (red) in the AcGFP1 and Neurod4-introduced groups at 5 DPI. High-magnification images of left panels are shown in the right panels. Arrowheads indicate Neurod4-introduced (AcGFP-positive) and immature (DCX-positive) neurons.(B) Representative images of tauAcGFP1 (green), NeuN (red), and DAPI (blue) in the tauAcGFP1 and Neurod4-introduced groups at 7 and 42 DPI. High-magnification images of left panels are shown in right panels. Arrowheads indicate Neurod4-introduced (AcGFP-positive) and mature (NeuN-positive) neurons. The graphs show the percentage of NeuN-positive cells in GFP-positive cells around the CC in the tauAcGFP1 and Neurod4-introduced group at 7 and 42 DPI. Statistical analysis was performed using student's-t test: ∗p < 0.05 and ∗∗∗p < 0.0005. Data represent the mean ± S.D. DCX, doublecortin.
	Figure 4. Promotion of the differentiation of excitatory and inhibitory neurons by Neurod4 expression in activated neural stem cells(A) Representative images depicting in situ hybridization (ISH) analysis of Slc17a6 (purple), the mRNA for the excitatory neuronal marker VGlut2, and Slc6a5 (red), the mRNA for the inhibitory neuronal marker GlyT2, in AcGFP1-positive cells expressing Neurod4 at 42 DPI.(B) Relative quantification of mRNA expression levels of neuronal subtype marker genes, Slc17a6 and Slc6a5 in AcGFP1 and Neurod4-expressing spinal cords at 42 DPI. Statistical analysis was performed using student's-t test: ∗p < 0.05 and ∗∗p < 0.005. Data represent the mean ± S.D.(C) The percentage of neuronal subtypes differentiated from Neurod4-introduced GFP-positive cells. The number of positive cells was calculated using the H-score of RNAscope as the semi-quantitative expression level.(D) Representative images of ChAT (red) in Neurod4-introduced AcGFP1-positive cells (green) in the spinal cord of the Neurod4-introduced group mice at 42 DPI. Images are zoomed up to right panels in rectangles of left panels. Arrowheads indicate the expression of ChAT in Neurod4-introduced AcGFP1-positive cells around CC. ChAT, choline acetyltransferase.
	Figure 5. Projections into motor neurons of Neurod4-introduced excitatory and inhibitory neurons(A) Representative images of cell soma of Slc17a6-expressing (excitatory) Neurod4-introduced Syp-AcGFP1-expressing neurons at the dorsal portion of the gray matter, laminae (IV–V) and excitatory synapses in the motor neurons at the epicenter and at levels L2-L5 of the spinal cord. Arrows indicate the cell soma labeled by Slc17a6 (excitatory) (purple) in Neurod4-introduced cells (Syp-AcGFP1, green), and arrowheads indicate synapses labeled by presynaptic markers from Neurod4-introduced cells (Syp-AcGFP1, green), and the excitatory postsynaptic marker (PSD-95, red)-positive in the motor neurons at the epicenter and at levels L2-L5 of the spinal cord.(B) Schematic cartoon of projection of newly formed relay neurons from the epicenter to motor neurons at the levels L2-L5 of the spinal cord.(C) Representative images of the cell soma of Slc6a5-expressing (inhibitory) Neurod4-introduced Syp-AcGFP1-expressing neurons at the intermediate portion of the gray matter, (laminae V–VI) and inhibitory synapses in the motor neurons at the epicenter and levels L2-L5 of the spinal cord. Arrows indicate the cell soma labeled by Slc6a5 (inhibitory) (red) from Neurod4-introduced cells (Syp-AcGFP1, green), and arrowheads indicate synapses labeled by presynaptic markers from Neurod4-introduced cells (Syp-AcGFP1, green), and the inhibitory postsynaptic marker (GlyR, red)-positive in the motor neurons at the epicenter and levels L2-L5 of the spinal cord.(D) Schematic cartoon of projection of newly formed inhibitory neuron from epicenter to motor neurons at the epicenter. MN, motor neuron.See also Figure S4.
	Figure 6. Glial scar suppression and axonal tracing from projection neurons of M1 cortex beyond injured region of spinal cord after recovery(A) Representative images of GFAP (red) and DAPI (blue) in tauAcGFP1 and Neurod4-introduced cells at 7 and 42 DPI. Arrowheads indicate the expression of GFAP protein.(B) The percentage of GFAP-expressing astrocytes relative to DAPI-labeled cells in the injured spinal cord of tauAcGFP1 and Neurod4-introduced mice at 7 and 42 DPI. Statistical analysis was performed using student's-t test: ∗∗p < 0.005; and N.S. = not significant. Data represent the mean ± S.D.(C) Tissue clearing images of the axons in the corticospinal tract of the post-recovery spinal cord show pal-mKate2-labeled axons (red) in the control group (left, AcGFP1-introduced) and the Neurod4-introduced group (right). White broken line indicates the border of the injury site.(D) Axial images of the axons in the corticospinal tract at the rostral, epicenter and caudal regions are represented as a red dot. High-magnification images of upper panels are shown in lower panels in each sample. A yellow broken line indicates the border between white matter and gray matter. Arrows indicate the axons through corticospinal tract in the caudal region.See also Figures S3 and S5.
	Figure 7. Synaptic formation and functional recovery by Neurod4 after SCI(A) Excitatory synapses detected with PSD-95 antibody (light blue) and presynaptic marker (Syp-mKate2) (red) around AcGFP1-expressing cells at the dorsal portion of gray matter, laminae IV–V, at the epicenter of AcGFP1 or Neurod4-introduced group at 42 DPI. Arrowheads indicate the excitatory synapses labeled by a postsynaptic marker (PSD-95) (light blue) and presynaptic marker (Syp-mKate2) (red).(B) Excitatory synapses detected with PSD-95 antibody and presynaptic marker (Syp-mKate2) around ChAT-expressing cells at levels L2-L5 of the spinal cord of AcGFP1, Neurod4-introduced or sham-operated group at 42 DPI. The left panels show the motor neurons stained with anti-ChAT (light blue) antibody in the ventral horn at levels L2-L5 of the spinal cord beyond the injury site in the AcGFP1 and Neurod4-introduced mice at 42 DPI, respectively. Yellow arrowheads indicate ChAT-neurons surrounded with presynaptic markers, Syp-mKate2 (red). The right panels show excitatory synapses detected with PSD-95 (light blue) and Syp-mKate2 (red) in the ventral horn at levels L2-L5 of the spinal cord beyond the injury site in the AcGFP1 and Neurod4-transduced mice at 42 DPI. The lower panel shows excitatory synapses detected with PSD-95 antibody and presynaptic marker (Syp-mKate2) at levels L2-L5 of the spinal cord of sham-operated mice. Arrowheads and arrows indicate functional excitatory synapses and clusters of synapses, respectively.(C) The number of functional synapses in an area (1013 mm2) in the ventral horn beyond the injury site at levels L2-L5 of the spinal cord of mice at 42 DPI (3 mice per group). Statistical analysis was performed using an unpaired t test: ∗∗∗∗p < 0.00005. Data represent the mean ± S.D.(D) Improvement in hindlimb locomotor function was evaluated using the Basso Mouse Scale (BMS) in Neurod4 and AcGFP1-introduced mice for 6 weeks (5 mice per group). Statistical analysis was performed using student's t-test: ∗p < 0.05, ∗∗p < 0.01. Data represent the mean ± S.E.M.(E) Summarized schematic cartoon. Neurod4 introduction differentiates the ependymal-derived neural stem cells into VGlut2-positive-excitatory, GlyT2-positive inhibitory, and ChAT-positive motor neurons. Descending fibers from the M1 cortex synapse with these neurons and motor neurons at levels L2-L5 of the spinal cord, respectively. The VGlut2-positive excitatory neurons relay to motor neurons at levels L2-L5 of the spinal cord.

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