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Fig. 3 Scgn deficiency impairs neuronal development, and R79W mutation is functionally impaired. a Bright-field images of zebrafish embryos classified in three groups based on head and body morphology at 48 hpf (hours post fertilization). Microcephalic: the brain size is moderately reduced; delayed: fish development is severely delayed, and the brain size is significantly reduced. Scale bar, 100 μm. b Percentage of embryos in each group as defined in A. N stands for the number of embryos examined from three independent experiments. +/+: scgn+/+; +/−: scgn+/−; −/−: scgn−/−. c Relative length of eyes of embryos at 48 hpf. Experiments were repeated three times. ****p < 0.0001, **p < 0.01. P-values were calculated using one-way ANOVA, Tukey’s multiple comparisons test. d Relative distance between a pair of eyes of embryos at 48 hpf. Experiments were repeated three times. ***p < 0.001, ns not significant. P-values were calculated using one-way ANOVA, Tukey’s multiple comparisons test. e Classification of zebrafish embryos based on the expression level of HuC (elavl3) at 48 hpf. Reduce: the brain size is moderately decreased; Extremely reduced, the brain size is significantly reduced.(Top) Lateral views; (Bottom) dorsal views. f Percentage of embryos in each group as defined in E. N stands for the number of embryos used for statistical analysis. All experiments were performed in three repetitions. g Multispecies alignment of SCGN protein sequences, Sequence of the ASD0010 proband is included as a comparison. h Detailed view of zebrafish SCGN Q75 (yellow stick, equivalent to human R79) and its surrounding residues (PDB:6JLH). Residues located within 4 Å of Q75 are shown in stick representation. i Hb9 (green) expression in Tg [Hb9: GFP] transgenic zebrafish. Enlarged views are shown. Ctl: control MO injection; MO: scgn MO injection; MO + WT: co-injection of scgn MO and wide-type human scgn mRNA; MO + R79W: co-injection of scgn MO and mRNA encoding SCGN R79W; MO + R79Q: co-injection of scgn MO and mRNA encoding SCGN R79Q. All injections were performed at one-cell stage of the Tg [Hb9: GFP]ml2 transgenic zebrafish embryos. Scale bar: 20 μm. j Statistical results of the length of CaP axons in embryos were treated as in c. For each group, ~48 axons from 6 to 12 Tg [Hb9: GFP]ml2 transgenic zebrafish embryos are scored. Experiments were repeated three times. ****p < 0.001, ***p < 0.001, ns not significant. P-values were calculated using one-way ANOVA, Tukey’s multiple comparisons test. All experiments were performed in three repetitions