miRNA-9 regulates Mauthner-cell axon regeneration in vivo. a The phylogenetic tree with the maximum-likelihood algorithm of pri-miRNA-9-1 in humans, mice, rats, Xenopus, zebrafish, and Desmodus rotundus. The number represents the degree of homology, and miRNA-9-1 in zebrafish is highly homologous to miRNA-9-1 in humans. b Sequence alignment of pri-miRNA-9-1 in humans, mice, rats, Xenopus, zebrafish and Desmodus rotundus. Sequences and names were obtained from miRbase. The nucleotide sequences marked in red and orange are the mature miRNA-9, where red is the preferred strand and orange is the non-preferred strand. c Expression of miRNA-9-1 during 0-10 dpf development in Zebrafish. d Timeline of time points of electroporation, axotomy, and regeneration imaging. e Representative regeneration images of the M-cell axon with miRNA-9 overexpression. White asterisk, injury site; arrowhead, axon regeneration terminal; scale bar, 50 μm. f Construction of the miRNA-9 expression system. Plasmids express only mCherry served as the control vector. g miRNA-9 overexpression promotes M-cell axon regeneration (control: 313.3 ± 31.04 μm, n = 15 fish; miRNA-9 oe: 672.1 ± 38.87 μm, n = 15 fish). p < 0.0001. h Representative regeneration images of the M-cell axon with miRNA-9 sponge. Asterisk, injury site; arrowhead, axon regeneration terminal; scale bar, 50 μm. i Design of miRNA sponges. The construction of miRNA sponges was manipulated by inserting multiple miRNA binding sites in the 3′UTR of the mCherry. Plasmids express only mCherry served as the control vector. j miRNA-9 knockdown inhibits M-cell axon regeneration (control: 250.8 ± 29.49 μm, n = 14 fish; miRNA-9 sponge: 25.17 ± 21.51 μm, n = 14 fish). White asterisk: ablation point; arrowhead, axon regeneration terminal. scale bar, 50 μm. p < 0.0001. Assessed by unpaired t-test
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