Fig. 7.

Membrane-tethered Wnts are required for synaptogenesis. (A-C) Cortical neurons were transfected with FingR-PSD95-GFP to tag PSD95 endogenously. Control neurons were co-transfected with mem-mCherry and showed strong PSD95 puncta localising to protrusions (A), which was lost when neurons were instead co-transfected with memNotum (B) or when control cultures were incubated with soluble, recombinant NOTUM (C). (D) Puncta expression could be rescued in memNotum transfected neurons by pre-treatment with the NOTUM inhibitor LP-922056. (E,F) Overexpression of WNT7A (O.E. WNT7A) did not alter PSD95 localisation to protrusions (E), whereas recombinant WNT7A (Rec. WNT7A) increased protrusion number (F). (G) Quantification of protrusion number in each group. (H) Quantification of PSD95-positive protrusions in each group. (I-O) Quantification (I) of ectopic PSD95 puncta on dendritic arborisation. Neurons were co-stained for PSD95 and bassoon (BSN) after transfection with either mem-mCherry (J), memNotum (K), mem-mCherry+soluble Notum (L), memNotum+LP-922056 (M), mem-mCherry+O.E. WNT7A (N), or mem-mCherry+Rec. WNT7A (O). (P) Quantification of PSD95/BSN puncta on transfected neurons. Statistical significance was addressed using one-way ANOVA with Dunnett's multiple comparison tests to compare relevant controls within groups and an unpaired, two-tailed Student's t-test to compare specific combinations. *P<0.05; **P<0.01; ***P<0.005. Experiments were performed in biological triplicate, with at least five fields per group analysed.