Figure 2.

Localized Wnt3a Determines Axonal Formation and Positioning

(A) Schematic representation of the mode of action of Iwp-2. Iwp-2 blocks Porcupine-mediated lipid modification and consequently the secretion of Wnt proteins.

(B) Div0 neurons were either left untreated or treated with Iwp-2 (1 μM). Neurons were fixed at Div4 and stained for Wnt3a. Neurons were counterstained with Phalloidin (actin filaments; red) and DAPI (nuclei; blue) (upper panels: merged picture; lower panel: inverted gray-scale channel of the endogenous Wnt3a) (scale bar, 10 μm). Wnt3a accumulates peri-nuclear after treatment with Iwp-2 (upper panel), and the staining is decreased in the axon (lower panel) (scale bar, 10 μm).

(C) Neurons were either left untreated or treated at Div0 with Iwp-2 alone, Iwp-2 (1 μM) + Wnt3a (40 ng/mL), or Wnt3a alone. At Div2 neurons were fixed and stained for Tau and counterstained with DAPI. Upper panel: merged staining of Tau proteins (green) and nuclei (DAPI; blue). Lower panel: inverted gray scale of intensity of the antibody staining against Tau. Experimental conditions from left to right: untreated neurons, neurons treated with Iwp-2 alone, Iwp-2 in combination with recombinant WNT3a, and recombinant WNT3a alone. Pictures show representative results of at least three independent experiments (scale bar, 10 μm). Red arrowheads indicate multiple axons; blue arrowheads indicate Wnt retention of Tau in the cell body.

(D) Graph representing the percentage of cells with axonal localization of Tau in each quantified picture normalized to the number of nuclei present in the analyzed field (***p < 0.005, n = 20, mean (S.E.M) values are shown).

(E) Div0 neurons were seeded on a micro-patterned coverslip containing either a pattern of fluorescent BSA (control) or a pattern containing a mix of recombinant Wnt3a protein with BSA. The patterns are indicated by the white boxes. Neurons were fixed on the coverslip at Div2 and stained for Tau (green), and nuclei were counterstained with DAPI (blue). Upper panel: representative pictures of neurons on the control pattern. A montage of single neurons was made with ImageJ. Lower panel: representative pictures of neurons on the Wnt3a pattern. Pictures show representative results of at least three independent experiments (scale bar, 10 μm).

(F) Axon positioning of the neurons in (E) were divided into three categories: no effect, axon specification, and axon specification and guidance. Neurons are stained for Tau (green) and nuclei counterstained with DAPI (blue).

(G) Percentage of neurons belonging to different categories on BSA or Wnt3a + BSA patterned coverslips (n = 30, ***p < 0.0001, mean (S.E.M) values are shown).