Figure S5.
EVL is required for DF morphogenesis, and influences dendritic spine plasticity. (A) Immunofluorescence labeling of primary cortical neurons derived from wild-type (WT) or EVL knockout (KO) mice at indicated days in vitro. β-III-tubulin labeling shows overall neurite and early axon morphology, while MAP2 specifically labels dendrites. Scale bar = 50 µm. (B–E) Quantification of neurite morphogenesis in cortical neurons derived from wild-type or EVL knockout mice at indicated days in vitro. (B) Number of primary neurites originating from the soma. (C) Number of higher-order branches (secondary or higher). (D) Early axon length. The presumptive axon was defined as a primary neurite with a length greater than three times longer than the minor neurites. (E) Average length of primary neurites (excluding presumptive axons). Central line = median, dashed lines = interquartile range (IQR). (B, C, and E) Kruskal-Wallis test corrected for multiple comparisons; n = 77–99 total neurons, N = 3 biological replicates, and (D) Mann-Whitney test; n = 32–35 total neurons, N = 3 biological replicates. (F and G) Representative Western blot (F) and quantification of (G) protein lysates from cortical neurons derived from wild-type or EVL knockout mice at indicated days in vitro, demonstrating complete loss of EVL, and no compensatory upregulation of VASP or MENA high and low molecular weight isoforms. Mean ± SD. Unpaired t test; N = 3 biological replicates. (H). Scatterplot of average speed of DF tips, calculated as the average absolute tip displacement between successive timepoints. Gray shaded region indicates average speed less than 0.0128 µm/s (non-motile DFs). Median ± IQR. Mixed-effects model; n = 280–641 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. is not significant. See also Fig. 4, Video 3, and Video 4. Source data are available for this figure: SourceData FS5.

EVL is required for DF morphogenesis, and influences dendritic spine plasticity. (A) Immunofluorescence labeling of primary cortical neurons derived from wild-type (WT) or EVL knockout (KO) mice at indicated days in vitro. β-III-tubulin labeling shows overall neurite and early axon morphology, while MAP2 specifically labels dendrites. Scale bar = 50 µm. (B–E) Quantification of neurite morphogenesis in cortical neurons derived from wild-type or EVL knockout mice at indicated days in vitro. (B) Number of primary neurites originating from the soma. (C) Number of higher-order branches (secondary or higher). (D) Early axon length. The presumptive axon was defined as a primary neurite with a length greater than three times longer than the minor neurites. (E) Average length of primary neurites (excluding presumptive axons). Central line = median, dashed lines = interquartile range (IQR). (B, C, and E) Kruskal-Wallis test corrected for multiple comparisons; n = 77–99 total neurons, N = 3 biological replicates, and (D) Mann-Whitney test; n = 32–35 total neurons, N = 3 biological replicates. (F and G) Representative Western blot (F) and quantification of (G) protein lysates from cortical neurons derived from wild-type or EVL knockout mice at indicated days in vitro, demonstrating complete loss of EVL, and no compensatory upregulation of VASP or MENA high and low molecular weight isoforms. Mean ± SD. Unpaired t test; N = 3 biological replicates. (H). Scatterplot of average speed of DF tips, calculated as the average absolute tip displacement between successive timepoints. Gray shaded region indicates average speed less than 0.0128 µm/s (non-motile DFs). Median ± IQR. Mixed-effects model; n = 280–641 total DF from 4–6 neurons per biological replicate, N = 3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s. is not significant. See also Fig. 4, Video 3, and Video 4. Source data are available for this figure: SourceData FS5.

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