Figure 5.

GFRα1 Regulates PC Migration by Counteracting NCAM Function in the Developing Cerebellum

(A) Co-expression and interaction between GFRα1 and NCAM in developing PCs of the E12.5 cerebellum of wild-type (Gfra1^WT) and Gfra1 knockout (Gfra1^KO) embryos. Arrows denote unspecific profiles arising from blood vessel autofluorescence. Insets show higher magnification of boxed areas. PLA, proximity ligation assay; VZ, ventricular zone; RL, rhombic lip. Scale bar, 50 μm (25 μm for insets).

(B) Quantitative analysis of the proportion of PC progenitors that migrated from the VZ to the MZ in wild-type (Ncam^WT), heterozygous (Ncam^HET), and Ncam knockout (Ncam^KO) E14.5 embryos injected with BrdU at E12.5. Histogram shows average ± SEM. ^∗∗p < 0.01 versus WT (n = 9 mice, one-way Anova); #p < 0.05 versus WT (n = 6, Student’s t test).

(C) Relative cumulative frequency of Lhx5⁺ cells exiting E12.5 cerebellar explants from the indicated genotypes normalized to explant surface area. Ncam^KO (green; n = 172 explants) and Ncam^HET (red; n = 160) versus Ncam^WT (blue; n = 100) Kolmogorov–Smirnov test, p < 0.0001.

(D) Relative cumulative frequency of Lhx5⁺ cells exiting E12.5 cerebellar explants from wild-type and Ncam knockout embryos in the presence or absence of purified GFRα1-Fc protein coated on the nanofiber surface. Ncam^KO (green, n = 108), Ncam^WT+GFRα1-Fc (red, n = 79), and Ncam^KO+GFRα1-Fc (purple, n = 106) versus Ncam^WT Kolmogorov–Smirnov test, p < 0.0001.

(E) Representative sections from E14.5 wild-type cerebellum stained with anti-NCAM (green) and anti-polysialic acid (PSA, red) antibodies. VZ, ventricular zone; RL, rhombic lip. Scale bar, 100 μm.

(F) Relative cumulative frequency of Lhx5⁺ cells exiting wild-type E12.5 cerebellar explants in the presence or absence of EndoN (blue, n = 108; red, n = 120; Kolmogorov–Smirnov test, p = 0.007).

(G) Quantitative analysis of the proportion of PC progenitors that migrated from the VZ to the MZ in WT (n = 9), Ncam^HET (n = 9), Gfra1^KO (n = 7 mice) and compound Gfra1^KO;Ncam^HET (n = 5) E14.5 embryos injected with BrdU at E12.5. Histogram shows average ± SEM. ^∗∗p < 0.01; ^∗∗∗p < 0.0001.

(H) Relative cumulative frequency of Lhx5⁺ cells exiting E12.5 cerebellar explants from the indicated genotypes normalized to explant surface area. Gfra1^KO (green; n = 105 explants) versus WT (blue; n = 103) Kolmogorov–Smirnov test, p < 0.0001. Gfra1^KO; Ncam^HET (red; n = 104) versus Gfra1^KO (green) Kolmogorov–Smirnov test, p < 0.0001.

(I) Relative cumulative frequency of Lhx5⁺ cells from wild-type E12.5 cerebellar explants migrating on control nanofiber surface (blue, n = 162 explants) or surface coated with purified full-length GFRα1 (red, n = 143 explants) or with a purified GFRα1 protein lacking the N-terminal domain (Δ1) that mediates interaction with NCAM (green, n = 150 explants). Kolmogorov–Smirnov test, p = 0.0003 (blue versus red curves), p = 0.7229 (blue versus green curves).