Figure 6.

SCLIP inhibition at early stages promotes primitive process retraction of stage 1 fusiform PCs. A, PC morphology observed in vivo at E19. E19 rat cerebellar slices were immunostained for SCLIP (green) and CaBP (red). At E19, many PCs have reached their final destination in the developing cerebellum but still exhibit a bipolar morphology with a single primitive process (arrows) beside the axon. SCLIP is present in PCs and is particularly detected as a punctuate staining along the main process (arrows). Scale bar, 20 μm (confocal microscopy; z-stack projection). B, Lentiviral-mediated RNAi inhibits SCLIP expression in PCs of E19 cerebellar cultures. E19 cerebellar organotypic cultures were infected with Lv-shSCLIP just after plating and immunostained for GFP (green), SCLIP (red), and CaBP (blue) after 6 div. SCLIP labeling is no more detected in transduced PCs visualized by GFP (arrow), whereas it remains clearly visible in untransduced PCs (arrowhead). Scale bar, 20 μm. C, Morphologies of PCs at 6 div after infection of E19 cerebellar cultures with Lv-shCtrl or Lv-shSCLIP. E19 cerebellar organotypic cultures were infected just after plating and immunostained for GFP (green) and CaBP (red) after 6 div. Whereas PCs transduced with Lv-shCtrl (visualized by GFP) are mostly in stage 1 with a bipolar morphology like untransduced PCs (red; arrowheads), PCs transduced with Lv-shSCLIP exhibit a round morphology without any process beside the thin axon. The asterisk indicates the morphology of a stellate stage 2 PC. Scale bar, 20 μm. D, E, Quantified results of three combined independent experiments illustrating the effect of SCLIP inhibition on PC dendritic differentiation stages at 6 and 7 div. Whereas PCs of E19 cerebellar cultures transduced with Lv-shCtrl are mostly in stages 1 or 2 after 6 (D) and 7 (E) div, respectively, ∼88% of PCs transduced with Lv-shSCLIP are in a “round stage.” Values shown are mean ± SEM.