Fig 1: 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) ß-catenin. Arrowheads highlight GFAP+ cells. Scale bar: 10 µm. GFAP+ astrocytes in Wnt7a and Wnt7b injected mice had a significantly higher level of active ß-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 2: OPC-derived Wnt ligand Wnt7b is essential for astrocytic network development and neurological function in vivo. A) Construction of Wnt7b-flox mouse strain and the overall experiment scheme. B) Immunostaining of BLBP showed that Wnt7b cKO resulted in shrunk BLBP morphology but did not affect BLBP+ cell number at P7 and P10. C) Immunostaining of GFAP and Cx43 showed that Wnt7b cKO resulted in reduced astrocyte processes and astrocytic Cx43 expression. Scale bar: 50 µm. D) Western blot result of total hippocampal protein lysate confirmed the reduced Cx43 and GFAP expression in Wnt7b cKO. E) Immunostaining of Olig2 and NG2 reflected no significant change in the OPC number in the Wnt7b cKO. F) Immunostaining of c-Fos showed that Wnt7b cKO resulted in decreased c-Fos+ neurons. Scale bar: 100 µm. G) Open field experiments showed that Wnt7b cKO mice spent less time and traveled a shorter distance in the center area, while the total travel distance was not significantly altered. Data presented as mean ± SD unless stated otherwise. N = 9 mice for behavioral tests, 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: OPC-derived Wnt ligands Wnt7a/b promote astrocytic network development in vitro. A,B) Wnt ligand-treated primary astrocyte cultures were subjected to immunofluorescence staining of Cx43. Wnt7a and Wnt7b, individually or combined, were able to promote the expression of Cx43, and this effect was abolished by the Wnt signaling pathway inhibitor XAV939. Scale bar: 50 µm. C) Wnt ligand-treated primary astrocyte cultures were subjected to RT-qPCR to detect the expression of astrocyte functional genes. Wnt ligands, especially Wnt7a and Wnt7b, were able to promote the expression of Gfap, Cx43, Grin2c, Kcnj10, and Slc1a2, which was abolished by Wnt signaling pathway inhibitor XAV939. Axin2 and Notum were used as the positive control of Wnt signaling pathway activation. D) Wnt ligand-treated primary astrocyte cultures were subjected to the scratch assay. Wnt7a and Wnt7b treatment, individually or combined, was able to increase the dye coupling area, and this effect was abolished by the Wnt signaling pathway inhibitor XAV939. Scale bar: 50 µm. E) Heatmap of calcium imaging in Wnt ligand-treated primary astrocyte cultures. An increased number of Ca2+ wave peaks could be induced by Wnt7a/b treatment. Data presented as mean ± SEM, n = 68. Data presented as mean ± SD unless stated otherwise. N = 3 independent experiments. p-values are calculated using one-way ANOVA with multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. not significant. #comparison between Wnt7a/b and Wnt7a/b+XAV939 groups. # p < 0.05, # # p < 0.01, # # # p < 0.001.
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