Fig 2: Wnt7a/b replenishment rescues the astrocytic network abnormality in the parental isolation model. A) Demonstration of micro‐injection procedure. The observation site was highlighted by the magenta square. B) Immunostaining of GFAP and active (nonphosphorylated) β‐catenin. Arrowheads highlight GFAP+ cells. Scale bar: 10 µm. GFAP+ astrocytes in Wnt7a and Wnt7b injected mice had a significantly higher level of active β‐catenin compared to 0.9% NaCl injected control (Saline). C) Immunostaining of BLBP showed that the number of BLBP+ cells was not altered by Wnt7a and Wnt7b injection, while the branch number was increased. The injection site was highlighted by the dashed line. Scale bar: 1000 µm. D) Wnt7a and Wnt7b injected mouse displayed increased GFAP+ cell branches. E,F) Immunostaining of Cx43, Cx30, and GFAP showed increased expression of Cx43, Cx30 in Wnt7a and Wnt7b injected mouse hippocampus. Scale bar: 50 µm. Data presented as mean ± SD. N = 3 mice for immunostaining experiments. p‐values are calculated using unpaired t‐test. *p < 0.05, **p < 0.01, n.s. not significant.

Fig 3: M2 macrophages infiltration predominates in human IPF lung tissues. a The expression of iNOS (M1 macrophage marker) and CD206 (M2 macrophage marker) in IPF fibroblastic focus were examined by immunohistochemistry. Representative images are shown (n = 7). b The expression of collagen I, β-catenin, Wnt7a, and α-smooth muscle actin (α-SMA) in human IPF lung tissues was measured by western blotting. The expression levels were quantified with ImageJ (lower panels; n = 3). GAPDH was used as a loading control. Results are normalized to the expression of each individual protein in the control which is given a value of 1 and are expressed as means ± SD (*p < 0.05 vs. Control). c Expression of Wnt7a protein in CD206+ M2 macrophages was measured by immunofluorescence assay. Arrow indicates individual cells positive for both CD206 (green) and Wnt7a (red). Representative images are shown

Fig 4: Wnt gene expression are differentially regulated during corpus callosum remyelination and in microglia exposed to myelin. a, b Tissue dissection to evaluate Wnt3a, Wnt5a and Wnt7a gene expression in the demyelinated corpus callosum (day 35 p.i.), and during remyelination (day 42 p.i.) N = 6–10 mice per experimental group. Statistics: *p ≤ 0.05; **p ≤ 0.01 vs. Sham; #p ≤ 0.05; ##p ≤ 0.01 vs. TMEV day 35 p.i. (post-infection); ANOVA followed by Bonferroni’s post-hoc test. c, d In vitro analysis of Wnt5a and Wnt7a gene expression of microglia following the addition of purified myelin. N = 6 independent experiments with three replicates. Statistics: *p ≤ 0.05 vs. basal; Student’s t test

Fig 5: M1 microglia secrete Wnt5a, while M2c microglia secrete the Wnt7a protein. a OX-42 staining of microglial cells following 24 h of stimulation with polarization agents to obtain the M1, M2a, and M2c activation states. b, d Wnt5a and Wnt7a gene expression by microglia in different activation states after 6 and 24 h of polarization. c, e Wnt5a and Wnt7a protein secreted after 24 h of polarization, evaluated in the supernatant of the cultures by ELISA. N = 4 independent experiments with three replicates. Statistics: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs. Control; ANOVA followed by Bonferroni’s post-hoc test. Scale bar: 25 μm