Wnt5a secreted by somatic cells suppresses canonical Wnt signaling in PGCs. (A) n-βcat (red) in E9.5 PGCs cultured for 20 h in isolation (ISO) or with exogenous WNT5a (250 ng/ml). Bar, 10 µm. Quantification shows reduced n-βcat in individual PGCs in presence of WNT5a. Each dot represents a single PGC; bars indicate the mean and boxes denote middle 50% of data points. n = 3 experiments, 244–254 cells per condition; ***, P < 0.001 by Student’s t test. (B) Expression of candidate Wnt targets by qRT-PCR in ex vivo cultured E9.5 PGCs from A decreases in the presence of WNT5a. Results are normalized to the mean of Gapdh/Rpl7 and presented relative to ISO control. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ⁺, P = 0.052 by Student’s t test; error bars indicate standard error of the mean. (C) E-cadherin (Cdh1, magenta) immunofluorescence in E9.5 PGCs cultured for 20 h. Images are representative of mean values. Bar, 10 µm. Quantification confirms reduced expression of E-CADHERIN in the presence of WNT5a. n = 3 experiments, 212–231 cells per condition; ***, P < 0.001 by Student’s t test. (D) n-βcat (red) in E9.5 PGCs (green) cultured 20 h in isolation, with WT MEFs, Wnt5a^−/− MEFs, or Wnt5a^−/− MEFs plus exogenous WNT5a. Bar, 10 µm. Quantification of n-βcat in individual PGCs shows absence of suppression by Wnt5a^−/− MEFs that is restored with exogenous WNT5a. n = 3 experiments; 86–104 cells each; ***, P < 0.001 by analysis of variance.