Fig. 1

Stages of neuron differentiation: cilia promote axon outgrowth. A Schematic illustration of neuron differentiation stages [36]: stage 1 – growth cone protrusions during neuron polarization; stage 2a – symmetric bipolar stage with microtubule invasion into growth cones, first neurite extension; stage 2b – symmetric bipolar stage with microtubule bundles consolidation, further neurite extension; stage 3 – the emerging axon outgrows the opposite neurite breaking the bipolar symmetry; stages 4 and 5 – dendrites and dendritic spines formation, further maturation with formation of synapses. B-F Staging of human LUHMES neuron differentiation: immunocytochemistry detects nuclei (Hoechst staining), the cytoskeleton (Phalloidin stains F-actin), microtubule bundles (TUBB3) and emerging synapses (PSD95). G Neurons at differentiation stage 2a display a significantly larger diameter of the emerging axon than neurons at stages 2b and 3 (n = 20–26). H Axon elongation between differentiation stages 2a, 2b and 3 (n = 21–37). I Phalloidin staining is used to identify the cell body (dashed line) and the axon marker TRIM46 to recognize the initial segment of the axon (arrowhead and bracket), the diameter of which was used for measurements. Mean values are shown ± s.e.m. (G) and displayed as Box and Whisker plots (min to max) (H). The results are from two independent experiments including two technical replicates each. We conducted regular one-way ANOVA analyses with multiple comparisons (Bonferroni’s test) between groups. **p < 0.005; ***p < 0.0005; ****p < 0.0001