Fig. 2: DCX-KO neurons establish more branches through increased rates of initiation.

Images representative of 3 experiments, showing a neurite initiation (A), growth/shrinkage (B) and retraction (C) events in a CTRL0 neuron recorded after incubation with tubulin and actin binding dyes: SiR-tubulin (green) and Cellmask actin (magenta). Yellow arrowheads show actin protrusions where a new neurite is initiated. cyan arrowheads show the remains of a neurite retracting completely. Yellow arrows show growth events and cyan arrows show retraction events. These events were used for calculation of growth and retraction speed (D, E), by measuring neurite length and lifetime on kymographs. An example kymograph is shown in the bottom panel in (B) with brightfield and fluorescence imaging. D Distribution of neurite growth rate in CTRL0 and DCXKO1 neurons. E Distribution of neurite retraction rate in CTRL0 and DCXKO1 neurons. For (D, E), n = 26 CTRL0 and 38 DCXKO1 events, from 2 independent experiments. Statistical analysis was performed using a Mann-Whitney U test: ns, nonsignificant. F Distribution of the number of neurite retraction events/min seen in CTRL0 and DCXKO1 neurons. Cells showing no retraction events were included in the analysis, but the points were not represented on the plot. G Distribution of the number of neurite initiation events/minute per neuron in CTRL0 and DCXKO1 neurons. Cells showing no initiation events were included in the analysis, but the points were not represented on the plot. H Number of neurites found per neuron in CTRL0 and DCXKO1 neurons. For (F–H), n = 68 CTRL0 and 93 DCXKO1 neurons from 2 independent experiments. Statistical analysis was performed using a Mann-Whitney U test: ns nonsignificant, *p < 0.05, ***p < 0.001. I Distribution of neurite density per neuron for CTRL0 and DCXKO1 neurons in fixed samples. J Distribution of neurite number per neuron for CTRL0 and DCXKO1 neurons in fixed samples. For (I, J), n = 323 CTRL0 and 351 DCXKO1 neurons from 3 independent experiments. Statistical analysis was performed using a Mann-Whitney U test: ***p < 0.001. p-values are detailed in Supplementary Table 1.