Figure 3.

Canonical Wnt Signaling Regulates Axonal Outgrowth

(A) Schematic representation of a two-compartment microfluidic chamber (MFC) used in the study (upper scheme). The lower scheme represents the imaged area (1) with the two compartments, in one compartment (proximal) neurons are seeded in the other compartment (distal) either control-conditioned medium or Wnt3a-conditioned medium is added.

(B) Graph representing the number of neurons per 100 μm². Neurons were stained with DAPI, and the number of cells was determined by counting the nuclei. The graph shows the average cell number of three independent experiments (n > 50, n.s., not significant, mean (S.E.M.) values are shown).

(C) Neurons were fixed at Div3 and subjected to immunofluorescence staining using antibodies for LRP6 (magenta) and Tau (green). Picture shows LRP6 accumulation at the tip of the axonal growth cone. Left: merged picture of the LRP6 staining and Tau staining. Right: Inverted gray scale of the LRP6 staining. Pictures show representative results of three independent experiments (scale bar, 10 μm).

(D) Neurons in the MFC were fixed at Div3 and subjected to immunofluorescence staining using antibodies for Tau and either β-Catenin or Gsk3β. Left: merged picture of the β-Catenin (magenta) and Tau (green) staining. Middle left: Inverted gray scale of the β-Catenin staining. Middle right: merged picture of the Gsk3β (magenta) and Tau (green) staining. Right: inverted gray scale of the Gsk3β staining (scale bar, 10 μm).

(E) Div0 neurons were seeded in the proximal compartment of the microfluidic chamber; the distal compartment was filled with either control-conditioned medium (left) or Wnt3a-conditioned medium (right). Axons were allowed to grow for 3 days. Afterward, neurons were fixed, stained with antibodies against Tau, and nuclei were counterstained with DAPI. Pictures show representative results of at least three independent experiments (scale bar, 50 μm).

(F) The graph shows the average axonal length of the neurons described in (E) (n > 30, ***p < 0.005, mean (S.E.M.) values are shown).

(G) Schematic representation of the Dkk1-mediated inhibition of Wnt binding to its co-receptor Lrp6.

(H) Div0 neurons were seeded in the proximal compartment of the microfluidic chamber, the distal compartment was filled with either Wnt3a-conditioned medium (left) or Wnt3a-conditioned medium supplemented with Dkk1 (right). Axons were allowed to grow for 3 days. Afterward, neurons were fixed and stained with antibodies against Tau and nuclei were counterstained with DAPI. Pictures show representative results of at least three independent experiments (scale bar, 50 μm).

(I) The graph shows the average axonal length of the neurons described in (H) (n > 30, ***p < 0.005, mean (S.E.M.) values are shown).