Figure 7.

The impairment of migration of GCPs outside the EGL in Tis21 knock-out mice is rescued in organotypic slices by Tis21. A, Representative confocal images of cerebellar organotypic slices, showing BrdU⁺ GCPs inside or outside the EGL (whose boundaries are indicated by a white broken line). Slices, obtained from P7 mice of the indicated genotype, were pulse-labeled with BrdU for 6 h, cultured for additional 72 h, fixed, and immunostained with anti-BrdU antibody. Scale bars, 100 μm. B, Percentage ratio between BrdU-positive cells localized outside the EGL, and the total number of BrdU-positive cells. Data are mean ± SEM from three experiments (3 mice per genotype were analyzed). ***p < 0.001, Student's t test. C, Test by a modified Boyden chamber of the intrinsic ability of purified GCPs to migrate. GFP⁺ GCPs isolated by FACS from P7 cerebellum of GFP mice (of the indicated genotype) were placed in the upper chamber, and cells migrating to the lower side of the membrane were counted. Mean cell number per field ± SEM were obtained from three separate experiments, counting 20 fields per well (at least 2 wells per experiment). The number of migrated Tis21-null GCPs, either Patched1 wild-type or heterozygous, was significantly reduced with respect to Tis21 wild-type GCPs. ***p < 0.01, Student's t test. D–G, In cerebellar slices, Tis21-retrovirus rescues the defective migration of Tis21-null GCPs. D, F, Representative confocal images of cerebellar slices, showing BrdU⁺ and Tomato-Tis21⁺ or Tomato-empty⁺ GCPs inside or outside the EGL. Slices, obtained from P7 Tis21 knock-out mice in Patched1 background, either Tis21^KO (D) or Ptch1^+/−/Tis21^KO (F), were infected with Tomato-Tis21 or Tomato-empty retrovirus, pulse-labeled with BrdU for 6 h, cultured for additional 72 h, fixed, and immunostained with anti-BrdU antibody. Scale bars, 100 μm. E, G, The percentage ratio between BrdU⁺/Tomato⁺ or BrdU⁺/Tomato⁻ cells outside the EGL, and the total number of BrdU⁺ cells was analyzed in slices from either Tis21^KO (E) or Ptch1^+/−/Tis21^KO mice (G), infected with Tomato-Tis21 or Tomato-empty retrovirus. The absence of effect in BrdU⁺/Tomato⁻ cells is a control of the specificity of infection. Data are mean ± SEM from three experiments (3 mice per genotype were analyzed). *p < 0.05, or **p < 0.01, Student's t test.