(a) Schematic representation of a microfluidic chamber. Neurons are plated into the left compartment (cell body compartment) and extend their axons through 450-µm-long microgrooves into the right compartment (axonal compartment). DRG neurons were used in these experiments since their axons are long enough to grow through the grooves, while dorsal spinal commissural (DSC) axons typically are too short to cross into the axonal compartment (U.H., S.R.J., data not shown). (b) Dissociated DRGs were cultured for 4 days in microfluidic chambers. Application of the protein translation inhibitors cycloheximide or rapamycin to the axonal compartment does not significantly change the basal growth rates (one-way ANOVA, P=0.7544), but completely abolishes NGF-induced axon growth. *P<0.05, n = 50 to 120 axons from 4 independent experiments per condition.

(a) Schematic representation of the spinal cord electroporation. A segment of E11 rat spinal cord is placed between the electrodes with the dorsal part facing the anode. Commissural neurons in the dorsal spinal cord are preferentially transfected. (b and c) E11 spinal cords 48 h after electroporation with pUB-GFP alone (b) or pUb-GFP with control siRNA (c). Most axons have reached the midline and turned anteriorly (arrow heads). (d and e) Knockdown of PAR3 and PAR6 mRNA greatly reduces the number of axons reaching the midline. Scale bar, 100 µm. (f) Quantification of the commissural axon growth reveals significantly stunted growth upon PAR3 or PAR6 siRNA transfection (Chi-square, P<0.001 and P=0.0311, respectively). (g and h) Initial segments of transfected axons were traced and their angle to the longitudinal axis of the spinal cord measured. siRNAs transfection does not change directionality as measured by a comparison of the variances of the angles (Bartlett’s test, P=0.1157). (i–l) Formation of the polarity complex is required for NGF and netrin-1 signalling. DRG (i and k) and DSC (j and l) explants were transduced on DIV3 and an axon outgrowth assay was performed 24 h later. (i, j) Axonal overexpression of EGFP or protein fragments that interrupt binding of PAR3 to PAR6 (PAR3: 246–363), PAR3 to aPKCζ (PAR3: 712–936), or PAR6 to aPKCζ (PAR6: 1–125), or overexpression of a kinase dead version of aPKCζ (aPKCζ KD), or pharmacological inhibition of aPKCζ (aPKCζ PSI) does not significantly decrease the basal axonal growth rate (one-way ANOVA, DRG: P=0.085, DSC: P=0.3764). (k, l) Change in axon growth rates relative to basal growth after stimulation with NGF or netrin-1. Overexpression of the dominant-negative proteins or inhibition of aPKCζ significantly inhibits or even reverses NGF- or netrin-1-induced axon growth while. The reversal of the growth-stimulating effect of NGF seen with some of the dominant-negatives might be due to the unmasking of parallel, NGF-dependent pathway. *P<0.05, **P<0.01, ***P<0.001. n = 28 to 88 individual axons from three independent experiments per condition.

(a) FISH on axons transfected with siRNAs. Dissociated DRG neurons were grown in microfluidic chambers, and siRNAs were transfected in the axonal compartment only. Cells were fixed 24 h after transfection and labelled for PAR3 and lacZ mRNA. (b) Quantification of the FISH signal reveals that transfection of PAR3 siRNAs into axons does not affect the PAR3 mRNA levels in the cell body compartment (one-way ANOVA, P=0.6224) but significantly reduces axonal PAR3 mRNA signals compared to transfection with control siRNA. Labelling for lacZ on control siRNA transfected axons is used to define the intensity of background labelling. *P<0.05, **P<0.01. n = 5 optical fields with 10 axonal segments each. (c) Axon outgrowth assays on axons transfected with PAR3 siRNA. DRG neurons were grown in microfluidic chambers and transfected with siRNA as above. Application of NGF to the axonal compartment induced axons growth in not transfected and control siRNA transfected axons while transfection with either PAR3 siRNA completely abolished the growth effect of NGF. Locations of the growth cones at 0 min and 60 min are indicated by green down () and red up triangles (), respectively. (d) Quantification of experiment in (c). The basal growth rate is not significantly affected by siRNA transfection (one-way ANOVA, P=0.0691). **P<0.01, *P<0.05. n = 20 to 115 axons per condition. Scale bars, 20 µm. (e) Application of NGF causes an increase in immunofluorescence staining intensity for PAR3 in distal axons and growth cones. SiRNA-mediated knockdown of axonal PAR3 mRNA abolishes the effect of NGF on PAR3 protein levels within distal axons and growth cones. **P<0.01. n = 17 to 30 axons from three experiments per condition.

(a, b) Immunolabelling of DRG (b) and DSC (c) explants at 5 DIV for polarity complex proteins shows localization of PAR3, PAR6 and aPKCζ to growth cones. (c, d) Dissociated DRG neurons were transfected with siRNAs targeting PAR3 or PAR6 mRNA or a non-tageting control siRNA and immunolabelling against PAR3 (c) or PAR6 (d) was performed 48 h later. (e, f) Quantification of the average staining intensity for PAR3 and PAR6 normalized to tau-1 staining reveals that transfection with targeting siRNAs causes a significant decrease in staining intensities demonstrating the specificity of the PAR3 and PAR6 signal observed in axons and growth cones. t-test, PAR3, *P=0.0104; PAR6, ***P=0.005. n = 13 to 21 axons per condition. Scale bar, 20 µm.

(a) FISH using digoxigenin-labelled riboprobes directed against lacZ and PAR3 transcripts. PAR3 transcripts are detected in axons and growth cones of DRG neurons. (b, c) Quantification of FISH signals for lacZ, RhoA, PAR3, PAR6, and aPKCζ transcripts in axons and growth cones of both DRG (b) and DSC (c) explants. The FISH signal for RhoA and PAR3 mRNA is significantly stronger than the inherent background defined by the intensity of labelling for lacZ. *P<0.05, **P<0.01, ***P<0.001. n = 5 optical fields with several axons each per condition. (d) β-actin and PAR3 mRNAs were detected by RT-PCR in DRG axonal lysates of harvested from microfluidic chambers while γ-actin, PAR6, and aPKCζ transcripts were not detected in axonal preparations. All transcripts were detected in cell body lysates. A, axonal lysate; C, cell body lysate. (e) Axons of DRG explants were severed from their cell bodies and incubated for 1 h with vehicle or 1 µM cycloheximide (CHX) and 100 ng ml⁻¹ NGF. The axons were fixed and immunolabeled with antibodies against PAR3 and tau-1. Application of NGF to severed axons leads to a translation-dependent increase in staining intensity for PAR3. (f) Quantification of the PAR3 staining intensity in a reveals a significant, cycloheximide-sensitive increase upon NGF stimulation (one-way ANOVA with Dunett’s post-test, **P<0.01. n = 5 to 10 axons per condition), while tau-1 staining intensity is not changed (P=0.1557). (g) DRG explants were transduced with PAR3 reporter pseudovirus on DIV3, and phase contrast and fluorescent images were taken after 24 h. Fluorescence images are shown in inverted contrast to more readily visualize puncta. Few puncta representing translation of the reporter are seen in axons of neurons expressing the PAR3 reporter at the beginning of the experiment. NGF stimulation (100 ng ml⁻¹) greatly increases the number and intensity of puncta. Application of cycloheximide leads to the complete absence of puncta both under baseline conditions and after NGF stimulation. Scale bars, 25 µm.