Enrichment of Wnt3a at the Growing Axon and a Local Activation of the Pathway Regulate Axonal Formation
(A) Endogenous Wnt3a expression at stages 2, 3, and 4 of neuronal development. Neurons were fixed at the indicated time points and stained with an antibody against Wnt3a (magenta). Neurons were counterstained with Phalloidin (actin filaments; green) and DAPI (nuclei; blue) (left side: merged picture; right side: inverted gray-scale channel of the endogenous Wnt3a expression [scale bar, 10 μm]).
(B) Left side: Schematic representation of a Wnt signalosome. Wnt binding induces clustering of Wnt ligands and its receptors Frizzled and LRP6. Right side: Neurons were transfected with Lrp6-GFP at Div3, fixed at Div4, and stained for the endogenous Wnt3a. Arrows indicate clustering and co-localization of Lrp6 (green) with Wnt3a (magenta) at the axon (scale bar, 10 μm).
(C) Gsk3β inactivation in the axon of stage 4 neurons. Neurons were fixed at stage 4 and subjected to an antibody staining against an inactivated form of Gsk3β (phospho-Gsk3β magenta). Neurons were counterstained with Phalloidin (actin filaments; green) and DAPI (nuclei; blue) (left side: merged picture; right side: inverted gray-scale channel of the phospho-Gsk3β signal) (scale bar, 10 μm).
(D) Neurons were co-transfected with GFP (to identify transfected neurons) and either an empty vector as a control (left panels) or an expression vector containing Wnt3 (right panels) at Div0. Neurons were either left untreated (upper panel) or treated with Taxol (10 nM, lower panel) at Div1. Neurons were fixed at Div 4 and stained for AnkG. Pictures on the left side show an overlay of GFP (green), AnkG (axonal initial segment; red), and DAPI (nuclei; blue). Pictures on the right show the inverted gray scale of the AnkG staining (scale bar, 10 μm). Red arrowheads indicate axons.
(E) Bar graph representing the average number of axons per neuron, determined by the number of axonal initial segments per cell (n = 20 ***p < 0.0001, mean (S.E.M) values are shown).