Figure 1

GNP proliferation in the cerebellum in vivo and in vitro . P7 WT and KO mice were injected with BrdU and killed 2 h later to perform BrdU LI analysis in the EGL. (A) Low magnification (4×) images of 5-μm sagittal sections from WT and KO cerebellar vermis are shown. As previously described [14, 16], the KO cerebellum exhibits an overall smaller size. Arrows point to lobule IV. (B) At higher magnification, representative vermis sections show the middle top portion of the folium of lobule IV. Overall tissue cytoarchitecture appeared similar, and BrdU labeled GNPs were localized normally in both genotypes. Arrows denote proliferating GNPs in the outer EGL immunostained for S-phase marker BrdU; arrowheads denote post-mitotic GNPs in the inner EGL. Insets show cells in the EGL. Bar = 50 μm; inset bar = 10 μm. (C) When assessed across the entire extent of the cerebellum, the BrdU LI was moderately increased in the KO. (D) There was a greater increase in the KO LI when assessed within the cerebellar vermis. N = 4-5 animals per genotype; 8–14 sections were counted per entire cerebellum; 4–6 sections were counted per vermis region. (E) Twenty-four-hour cultures of WT and KO GNPs in control media are shown after fixation and BrdU immunocytochemisty. Upper panels show isolated GNPs under phase microscopy, whereas lower panels reveal brightfield images. Arrows point to cells in phase that exhibit BrdU immunostaining in lower panels. Bar = 10 μm. (F) The GNP BrdU LI in vitro was two-fold greater in the KO compared to WT cells. For each experiment, GNPs were isolated from groups of 4–6 mice per genotype. N = 3 culture dishes per genotype per experiment, and three experiments were performed. EGL: External germinal layer; ML: molecular Layer; PCL: Purkinje cell layer; IGL: inner granule layer. t-test: *p ≤ 0.05; **p ≤ 0.01.