Increased nuclear β-catenin levels in PGCs during their migratory progression and perturbation in Ror2Y324C PGCs. (A) Nuclear β-catenin (n-βcat, red) immunofluorescence in transverse histological sections of an E9.75 WT embryo treated with Ficin enzyme to disrupt E-cadherin/β-catenin membrane staining. The hindgut (hg), neural tube (nt), and mesonephric duct (mes) are indicated. Bar, 30 µm. (i) PGCs are identified by expression of Oct4-ΔPE-GFP (green; white arrows in A). Bar, 30 µm. (ii) Pseudocoloring (rainbow) indicates individually selected Oct4-ΔPE-GFP+ PGCs for quantitative measurement of n-βcat. (iii) Pseudocoloring (rainbow) indicates DAPI-selected nuclei of all cells in the field (>600 counted) used to obtain the mean intensity of n-βcat. (iv) Inset from box in A and i to exemplify differences in n-βcat levels in PGCs (dashed white lines) relative to the mean n-βcat levels in all nuclei in the field. Fold differences in staining intensity are indicated. Bar, 10 µm. (B) Quantification of n-βcat in all E9.5 Ror2Y324C PGCs relative to WT/het littermates shows an increase in accumulation of n-βcat. Each dot represents a single PGC; bars indicate the mean and boxes denote the middle 50% of data points. n = 100–134 cells from two embryos per group; **, P < 0.01 by Student’s t test. (C) Quantification of n-βcat in E9.5 PGCs from B by location. *, P = 0.045; †, P = 0.074 by Student’s t test. (D) Quantification of n-βcat in migratory PGCs at various anatomical locations in E9.5–11.5 embryos. n-βcat in PGCs is normalized to mean n-βcat in surrounding nuclei as described in A and shown relative to the hindgut. Each dot represents a single PGC, and bars indicate the mean ± standard deviation. The dotted line indicates a trend of increasing n-βcat with migratory location. n = 25–203 cells per location from ten mixed CD1 embryos. R2 = 0.97; P = 0.002 by regression analysis; P = 0.009 by analysis of variance; **, P = 0.009 by Student’s t test.
