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First published online January 10, 2008
doi: 10.1242/10.1242/jcs.023143

Journal of Cell Science 121, 178-185 (2008)
Published by The Company of Biologists 2008
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Pyramidal neuron polarity axis is defined at the bipolar stage

Froylan Calderon de Anda1,*, Annette Gärtner2,*,{ddagger}, Li-Huei Tsai1,3 and Carlos G. Dotti2,{ddagger}

1 Department of Brain and Cognitive Sciences, Picower Institute for Learning and Memory, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building 46, Room 4235A, Cambridge, MA 02139, USA
2 VIB Department of Molecular and Developmental Genetics and Department of Human Genetics, Catholic University of Leuven, 3000 Leuven, Herestraat 49, Belgium
3 Howard Hughes Medical Institute, 77 Massachusetts Avenue, Building 46, Room 4235A, Cambridge, MA 02139, USA


Figure 1
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  		Fig. 1. Axon fate predisposition is present at the bipolar stage. (A) Neocortical slice of an E18 mouse brain that had been transfected with a cytoplasmic, fluorescent protein (Venus-EGFP) at E15 by in utero electroporation, showing migrating cells (lower panel). The upper panel shows the distribution of the axonal marker tau-1. The lower panel shows Venus-EGFP fluorescence merged with the tau-1 image. (B) Maximal intensity projection of a confocal z-stack of a cell in the upper intermediate zone (boxed in A). The GFP-positive cell has a typical bipolar morphology of migrating neurons (arrowheads indicate trailing neurite). The numbers at the left (1-7) mark the places along the neuron that were selected for y-z scans shown in C. (C) Views along the y-z plane. Arrowheads show a colocalization of GFP and tau-1. (D) GFP and tau-1 intensity-profiles from the selected places 1-7 confirm a colocalization of GFP and tau-1 immunoreactivity in the distal portion of the trailing process of the migrating neuron. The profile was obtained by drawing a line that crossed the middle of the GFP signal. Bar, 100 µm (for A) and 10 µm (for B,C).

 

Figure 2
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  		Fig. 2. Neurites and the axon are generated in a stereotypical sequence. (A,B) Time-lapse video microscopy of individual neurons showing that they extend the first two sprouts opposite to each other. (A) An example of a neuron in which the first sprout becomes the axon (arrowheads). (B) Example of a neuron that extended the axon (2; arrowheads) opposite to the first sprout (1). (C) Neurons migrating radially from an aggregate culture in Matrigel. The neurons exit the explant by extending one leading neurite (open arrowhead, 0'). The oppositely located trailing neurite is visible (solid arrowhead). (D) Migrating neurons in Matrigel cultures were fixed and stained with the axonal marker tau-1. Open arrow, leading neurite; arrowhead, trailing neurite. (E,F) Individual neurons were followed from the formation of the first bud (inset) until stage2. Stage-2 cells were fixed and the mean intensity of APC immunoreactivity measured in all neurite growth cones. In 70% of the observed neurons (33 cells) APC mean fluorescence was maximal either in the neurite appearing first (one example shown in E) or in the neurite appearing second (one example shown in F) neurite. Bars, 10 µm.

 

Figure 3
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  		Fig. 3. Neuronal cytoarchitecture is arranged in a graded bipolar manner. (A-C) MT repolymerization in early plated neurons (3-4 hours in culture). (A) Neurons were treated for 2-3 hours with nocodazole and MT repolymerization was visualized at different times after nocodazole washout in fixed cells. Between 2 and 5 minutes, MTs polymerize mainly towards the pole opposite to the nucleus where the centrosome is located (pole 1 in B). After 10 and 15 minutes, MTs polymerize still towards the opposite pole (pole 2 in B) but less to the sides (pole 3 and 4 in B). (B) Quantification of the amount of MTs in the different poles of the neurons after repolymerization as shown in A (n=19). Neurons were divided in populations in which MTs sprouted towards one pole (as in A 2' and 5') and those with bipolar-orientated MTs (as in A 10'and 15': two poles). (C) The same experiment as in A and B was performed in stage-3 neurons (n=11: 1 pole, n=9: 2 poles). (D) Live cell analysis of post-Golgi membrane traffic after labelling with BODIPY-ceramide. In a cell with three sprouts (1,2,3) membrane traffic is preferentially directed towards the largest lamellipodium (0',1), at later times also towards the opposite pole (2), followed by sporadic traffic to a third pole (3). (E) Quantification of mean intensities of BODIPY-ceramide fluorescence in neurites from cells with three neurites (n=15 cells). (F) The traffic of vesicles and membrane compartments in live cells into all neurites of polarized stage-3 neurons was imaged by phase-contrast microscopy. The number of membrane carriers travelling within 1 minute through a defined proximal and distal neurite segment (n=5 cells) was counted. (B,C,E,F) Statistical analysis: ANOVA test followed by Tukey's multiple comparison test ***P<0.001, **P<0.01, *P<0.05 comparing the values to the first column or second as indicated. (G) F-actin disruption using cytoD (addition of 2 µM cytoD at 0 minutes) in cells with three sprouts, in which the order of the appearance of the sprouts was monitored before (–180 minutes, 0 minutes). CytoD addition leads to the highest growth from the first (1) and second (2) sprout. (H) Neurons were transfected with centrin-1–GFP before plating and centrosome position was monitored after 24 hours in neurons in which initially the first sprout faced the centrosome. These data are presented in the frequency distribution (n=30) graph showing the movement of the centrosome in degrees, with respect its initial position. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Axon lesion leads to axon formation at the opposite pole. (A) A polarized neuron (0'; axon: arrowheads), the axon of which was cut (5', arrow), develops a new axon opposite to the transected one (24 hours, arrowheads). Axonal identity was confirmed by tau-1 immunoreactivity (right panel). (B) Frequency distribution representing the site of axon regeneration after axotomy (n=187). 0° is the position of the original axon. Bin size 20°. (C) Double axotomy: after the first transection (5', arrow) the new axon formed opposite (24 hours, arrowheads) to the first one (0'). 24 hours after cutting this new axon (24 hours, arrow), axonal regrowth occurred again from the site of the initial axon (48 hours, arrowheads). Axonal identity was confirmed by tau-1 immunoreactivity (right lower panel). In eight out of the 16 cells that survived the double axotomy, the new axon formed from the original pole (from the stump of the original axon) in four cells or from a neurite that was close to the original axon in the other four cells. In four of the remaining cells the new axon grew from the `second' pole and in the last four cells a new axon grew from a random position. Bars, 10 µm.

 

Figure 5
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  		Fig. 5. Axonal respecification is preceded by the reversion of membrane flow and does not involve rotation of the centrosome. (A) Live cell analysis of post-Golgi membrane traffic visualized by labelling with BODIPY-ceramide after recovery from axotomy (2-3 hours after cutting: arrow) shows that more traffic is directed towards the opposite side (open arrowheads) from the sectioned axon (original axon at 0': arrowheads, n=15). (B) Neurons were transfected with centrin-1–GFP to mark the centrosome position (open arrowhead), and the centrosome was observed before axotomy and after axon regrowth. Arrowheads, axons; arrow, axotomy; open arrowheads, centrin-1–GFP. Bars, 10 µm. (C) Quantification of the results in B: frequency distribution showing centrosome position after axotomy with respect to its original position when the axotomy was performed (bin size 10°; n=26 neurons).

 

Figure 6
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  		Fig. 6. Stages of neuronal polarity development. The scheme represents the different stages of neuronal polarity development of hippocampal neurons in vitro. See Discussion for details.

 

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