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

Heterozygote knockout of miRNA-9 did not affect M-cell axon regeneration and growth and development in zebrafish larvae. a Generation of miRNA-9^± and Tg (Tol-056); miRNA-9^+/− zebrafish. miRNA-9^−/− can cause embryonic lethality. b, c Heterozygote knockout of miRNA-9 did not affect M-cell axon regeneration (control: 383.4 ± 22.37 μm, n = 17 fish; miRNA-9^+/−: 390.1 ± 20.69 μm, n = 24 fish). White asterisk: ablation point; arrowhead, axon regeneration terminal. scale bar, 50 μm. p = 0.8283, assessed by unpaired t-test, ns, not significant. d, e Total lengths of M-cell axons from the cloaca to the end were not notably different among WT and heterozygous knockout larvae (control: 1464 ± 17.8 μm, n = 8 fish; miRNA-9^+/−: 1491 ± 26.07 μm, n = 8 fish). White asterisk: ablation point; arrowhead, axon regeneration terminal. scale bar, 50 μm. p = 0.4173, assessed by unpaired t-test, ns, not significant. f, g Trajectory diagrams and motion distance statistics of free swimming within 1 h of miRNA-9^+/− (control: 584.6 ± 34.03 cm, n = 24 fish; miRNA-9^+/−: 545.3 ± 30.90 cm, n = 24 fish). p = 0.3973. Assessed by unpaired t-test. h, i Representative images of larvae from the wildtype and the miRNA-9^+/− at 6 dpf (scale bar, 500 μm), and measured total body length from 4 to 6 dpf (4 dpf: control: 3.68 ± 0.0226 mm; miRNA-9^+/−: 3.730 ± 0.0217 mm, p = 0.1189, n = 15 fish; 5 dpf: control: 3.993 ± 0.0252 mm; miRNA-9^+/−: 3.984 ± 0.0326 mm, p = 0.8324, n = 15 fish; 6 dpf: control: 4.082 ± 0.0192 mm; miRNA-9^+/−: 4.098 ± 0.0293 mm, p = 0.6459, n = 15 fish). Assessed by two-way ANOVA. ns, not significant

Sequence alignment and the EGFP sensor assay show that her6 is the downstream target of miRNA-9. a Schematic representation of the interaction of miRNA-9 with her6 and sequence comparison of miRNA-9, her6 3ʹUTR, and her6 3ʹUTR mutation (within the 2–7nt mutated). The zebrafish miRNA-9 mature sequence is shown in red, the seed sequence in yellow, and the mutant nucleotide sequence in blue. The homology of her6 seed sequences in humans, mice, Xenopus, and zebrafish is also shown below. b, c EGFP-her6 3′UTR showed strong fluorescent signals when co-injected with non-sense duplex (as negative control), but failed to give fluorescent signals when co-injected with miRNA-9 duplex. mCherry mRNA was injected as a control. d, e EGFP-her6 3′UTR mut showed strong fluorescent signals both in the non-sense duplex and miRNA-9 duplex. f, g The EGFP (f) and mCherry (g) fluorescence was expressed as a percentage of the fluorescent signal observed from the negative control with the EGFP-her6 3′UTR. p < 0.0001 in EGFP fluorescence, p = 0.3404 in mCherry fluorescence, assessed by t-test. h, i The EGFP (h) and mCherry (i) fluorescence were expressed as a percentage of the fluorescent signal observed from the negative control with the EGFP-her6 3′UTR mut. p = 0.1070 in EGFP fluorescence, p = 0.9303 in mCherry fluorescence, assessed by t-test

Deletion of her6 facilitates M-cell axon regeneration and has no effect on growth and development in zebrafish larvae. a Generation of her6^−/− and Tg (Tol-056); her6^−/− zebrafish. b, c Homozygote knockout of her6 promoted M-cell axon regeneration (control: 378.8 ± 21.57 μm, n = 20 fish; her6^+/−: 476.6 ± 26.42 μm, n = 19 fish, p = 0.0066; her6^−/−: 691.4 ± 36.86 μm, n = 20 fish; p < 0.0001). White asterisk: ablation point; arrowhead, axon regeneration terminal. scale bar, 50 μm. Assessed by unpaired t-test. d, e Total lengths of M-cell axons from the cloaca to the end were not notably different among WT, heterozygous and homozygote larvae (control:1406 ± 23.34 μm, n = 8 fish; her6^+/−: 1458 ± 36.60 μm, n = 8 fish; her6^−/−: 1441 ± 20.15 μm, n = 8 fish, p = 0.4140). White asterisk: ablation point; arrowhead, axon regeneration terminal. scale bar, 50 μm. p = 0.4140, assessed by one-way ANOVA, ns, not significant. f, g Trajectory diagrams and motion distance statistics of free swimming within 1 h of her6^−/−(control: 645.8 ± 28.62 cm, n = 24 fish; her6^+/−: 668.5 ± 28.90 cm, n = 24 fish; her6^−/−: 654.2 ± 33.29 cm, n = 24 fish). p = 0.8674. Assessed by one-way ANOVA. ns, not significant. h, i Representative images of larvae from the wildtype, her6^+/− and her6^−/− at 6 dpf (scale bar, 500 μm), and measured total body length from 4 to 6 dpf (4 dpf: control: 3.807 ± 0.0193 mm, n = 15 fish; her6^+/−: 3.820 ± 0.0238 mm, n = 15 fish; her6^−/−: 3.831 ± 0.01201 mm, n = 15 fish, p = 0.6737; 5 dpf: control: 3.938 ± 0.0168 mm, n = 15 fish; her6^+/−: 3.958 ± 0.0174 mm, n = 15 fish; her6^−/−: 3.970 ± 0.0194 mm, n = 15 fish, p = 0.4588; 6 dpf: control: 4.066 ± 0.0185 mm, n = 15 fish; her6^+/−: 4.034 ± 0.0153 mm, n = 15 fish; her6^−/−: 4.098 ± 0.0251 mm, n = 15 fish, p = 0.0907). Assessed by two-way ANOVA, ns, not significant

her6 regulates Mauthner-cell axon regeneration in vivo. a Construction of the her6 expression system. Plasmids express only mCherry served as the control vector. b Quantitative RT-PCR analysis exhibited overexpression of her6 in 4 dpf zebrafish larvae by the vector-based her6 oe in vivo. p = 0.0012. Assessed by unpaired t-test. c, dher6 overexpression inhibits M-cell axon regeneration (control: 255.8 ± 19.62 μm, n = 16 fish; her6 oe: 19.18 ± 9.603 μm, n = 16 fish). White asterisk: ablation point; arrowhead, axon regeneration terminal. scale bar, 50 μm. p < 0.0001. Assessed by unpaired t-test. e Design of the her6 shRNA expression system based on the miR-30e backbone. Plasmids express only mCherry served as the control vector. f Quantitative RT-PCR analysis exhibited a reduction of her6 in 4 dpf zebrafish larvae by the vector-based her6 shRNA-11 in vivo. p = 0.0019. Assessed by unpaired t-test. g, h Decreased expression of her6 inhibits M-cell axon regeneration (control: 253 ± 26.51 μm n = 15 fish; her6 shRNA-11: 442.7 ± 47.6 μm, n = 15 fish). White asterisk: ablation point; arrowhead, axon regeneration terminal. scale bar, 50 μm. p = 0.0017. Assessed by unpaired t-test

Her6 regulates calcium activity in M-cells and affects axon regeneration in vivo.a The time axis showed the time-points of electroporation, axotomy, dynamic calcium imaging, and regeneration imaging. One of the pharmacological-treated groups was treated with PTZ for 48 h after injury and then imaging of calcium activity and regeneration was performed. b Diagram of the brain of zebrafish and the location of electrical stimulation in M-cells. c Representative examples of M-cell calcium activity after electrical stimulation in larvae labeled with NEMOf. The time after the stimulation (in seconds) is given in each frame. The color scale indicates fluorescence intensity (black: lowest; white: highest; Scale bar, 20 μm). d The peak amplitude of the calcium response traces in larvae that regulate her6 expression by plasmid or pharmacologically (control: 16.96 ± 3.388, n = 8 fish; her6 oe: 2.108 ± 0.4145, n = 6 fish; her6 shRNA-11: 266 ± 43.45, n = 6 fish; her6 oe + PTZ: 457.3 ± 51, n = 6 fish). Assessed by one-way ANOVA. e, f The calcium response elicited in larvae overexpressing her6 was much weaker than that in control larvae and the response elicited in larvae her6 shRNA was much stronger than that in control larvae. g The relationship between regeneration length and peak amplitude of calcium response has a significant positive correlation (r² = 0.7375, n = 26). h, i Altered her6 expression affects M-cells axon regeneration (control: 241.5 ± 36.38 μm, n = 8 fish; her6 oe: 44.38 ± 21.65 μm, n = 6 fish; her6 shRNA-11: 563.3 ± 114.2 μm, n = 6 fish; her6 oe + PTZ: 457.3 ± 51 μm, n = 6 fish). White asterisk: ablation point; arrowhead, axon regeneration terminal. scale bar, 50 μm. Assessed by one-way ANOVA

Axon regeneration induced by her6^−/− promotes recovery of motor function in zebrafish. a Diagram of the equipment to introduce and record the escape response. b Representative images of the initial position and maximal turn angle position from the control and her6^−/− zebrafish larvae in the uninjured and injured groups. The red line represents the direction of the current position. θ represents the maximal angle of rotation of the head. scale bar, 1 mm. c A series of images of escape response in the control and her6^−/− zebrafish larvae in the uninjured and injured groups. Time after the escape response (in milliseconds) is given in each frame. * represents the time for the maximum turning angle scale bar, 1 mm. d The maximum turn angle of escape in the her6^−/− larvae was significantly higher than that in the control group after injury. (control + uninjured: 131.8 ± 9.591°, n = 9 fish; her6^−/− + uninjured: 136.2 ± 5.193°, n = 9 fish; control + injured: 90.8 ± 2.569°, n = 8 fish; her6^−/− + injured: 116.7 ± 4.823°, n = 7 fish). Assessed by one-way ANOVA. e The time required to reach the maximum turning angle in the her6^−/− larvae was significantly shorter than that in the control group after injury. (control + uninjured: 9.778 ± 0.9969 ms, n = 9 fish; her6^−/− + uninjured: 10.78 ± 1.4320 ms, n = 9 fish; control + injured: 18.25 ± 0.5261 ms, n = 8 fish; her6^−/− + injured: 13.41 ± 0.5281 ms, n = 7 fish). Assessed by one-way ANOVA