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Files in this Data Supplement:
Fig. S1. (A) Migrating neuron (boxed) in the ventricular zone with a leading edge. (B) Enlargement of cell in A that is nestin negative (red arrowheads in lower panel) with the centrosome at the base of the leading edge (upper panel). Different confocal sections are shown. Bar, 10 µm.
Fig. S2. Neurons form the first two sprouts, visualized by their high F-actin content, mostly (in 88% of the cells) in an opposite orientation. Freshly plated neurons were fixed between 1 and 3 hours after plating and F-actin visualized with phalloidin-TRITC. Bar, 10 µm.
Fig. S3. Neurons were plated and observed by time-lapse video microscopy from the formation of the initial neurites to the time of axon formation. Shown here is the time just before axon formation occurs. The retractions and extensions of individual neurites were measured and quantified using a specially written plugin for ImageJ. We observed that a complementary extension (red arrowhead)-retraction behaviour between the first (1) and second (2) neurite is evident before the axon formation occurs. In all cases analyzed (n=6) the quotient between the numbers of extension for each neurite was calculated when a complementary behaviour occurred (one neurite extended, the other retracted). This analysis was calculated for the first three neurites comparing the first with the second, the first with the third, and the second with the third. A quotient of 1 expresses an equal occurrence of both neurites extending, in a certain number of complementary extension-retraction movements. If the quotient is different from 1 then one neurite is extending more, meanwhile the other is retracting more. Comparing these ratios for different neurite pairs suggests a competition between the first and second neurite because the quotient for this pair is close to 1 (neurite 1 ÷ neurite 2=0.94±0.04) whereas for the other pairs this quotient is different from 1 (neurite 1 ÷ neurite 3=1.78±0.67; neurite 2 ÷ neurite 3=1.59±0.26). Bar, 10 µm.
Fig. S4. Microtubules (MTs) repolymerize in a preferential bipolar orientation after recovery from nocodazole-induced depolymerization at stage 0 and 3. (A) For quantification of the orientation of MT sprouting, cells were divided in quadrants. Quadrant 1 corresponds to the pole opposite to the nucleus at the pole from which MTs sprout; quadrant 2 is the pole opposite to quadrant 1, quadrants 3 and 4 are perpendicular to quadrants 1 and 2. The mean intensity of the MT signal was measure in each quadrant and normalized for each cell. (B) One example of a stage-3 neuron 5 minutes after nocodazole washout (treatment with nocodazole: 7 µM, 2 hours) during which MT repolymerization occurred. Arrowheads show the significantly longest neurite, which is probably the axon. Inset: MTs were visualized with an antibody against α-tubulin (open arrowheads); the centrosome with a pericentrin antibody (arrow); and the nucleus with Hoechst. Bars, 10 µm.
Fig. S5. Neurons were transfected with centrin 1-GFP before plating. (A) Neurons in which initially the first sprout faces the centrosome were imaged and the position of the centrosome after 24 hours was visualized. (B) Centrosome position was followed in an individual neuron from the bipolar stage to stage 3. Bottom panel shows tau-1 staining to identify the axon. Bars, 10 µm.
Fig. S6. Application of cytoD resulted in the preferential growth of the first and the second sprouts, in a clear bipolar fashion. (A) F actin disruption using cytoD (2 µM, addition at 0 minutes) in a neuron with two sprouts, for which the order of their appearance was known (−180 minutes, 0 minutes). Notably after F-actin disruption the highest sprouting occurs from the first formed sprout (1, 480 minutes, lower panel: α-tubulin staining of the same neuron). (B) Quantification of the sprout area in A before and after cytoD addition. Bars, 10 µm.
Fig. S7. Par3 distribution in early fixed (2-3 hours after plating) hippocampal neurons. Par3 was detected by a rabbit IgG1 antibody (Upstate) and the Golgi visualized by a GM130 antibody (BD Biosciences). There is no evident polarization of Par3 towards the pole of the first or second sprout (see upper panel, neuron with one sprout; lower panels: neuron with two sprouts). Bars, 10 µm.
Movie 1. Bipolar arrangement of membrane traffic in stage-2 neurons. Neurons were transfected with a farnesylated EGFP, which labels all intra- and extracellular membranes. After 24 hours live cells were observed by epifluorescence microscopy in 1-second intervals. Membrane flow is arranged in a bipolar manner. The Golgi is well visible facing the first neurite, which later becomes the axon.
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