BrdU labeling reveals that early differentiating GCs have short primary cilia that subsequently disassemble. (A) P7 mice were injected with the thymidine analogue BrdU and cerebella were harvested after 12 or 48 h. BrdU incorporates into the DNA of cycling cells during the S phase. BrdU-labeled cells that began differentiating became P27^KIP positive and migrated deeper. (B) BrdU-labeled cells were classified by location in the developing cerebellum. The distribution of BrdU-labeled GCs was quantified 12 and 48 h after BrdU injection. (C) The percentages of total BrdU labeled cells in each layer that expressed P27^KIP 12 and 48 h after BrdU injection were quantified from three sections from two to three animals. Shapes denote biological replicates. (D) Sagittal sections of P7 mouse cerebella harvested 12 h after BrdU injection were immunostained with antibodies to BrdU (white), P27^KIP (yellow), ARL13B (magenta), and counterstained with Hoechst (blue in merge panel) before imaging with widefield microscopy. The approximate boundaries between layers are indicated in white and yellow. Arrows denote BrdU labeled P27^KIP positive cells. Scale bar: 10 μm. (E) BrdU-labeled ciliated cells in the inner and outer EGL were distinguished based on P27^KIP expression and cells at the inner edge of the EGL were directly adjacent to the ML. The measured lengths of cilia in these cells are plotted. n = number of cilia counted. (F) The percentages of BrdU-positive GCs with cilia in the outer EGL (P27^KIP negative), the inner EGL (P27^KIP positive), and at the inner edge (P27^KIP positive) were quantified 12 h after BrdU administration. (G) Sagittal sections of P7 mice cerebella harvested 48 h after BrdU injection were immunostained with antibodies to BrdU (white), P27^KIP (yellow), and ARL13B (magenta), and counter-stained with Hoechst (blue in merge panel) before imaging using widefield microscopy. The approximate boundaries between layers are indicated in yellow. Arrows denote BrdU labeled P27^KIP positive cells. Scale bar: 10 μm. (H) The length of each cilium in the indicated layers 48 h after BrdU labeling is plotted. EGL layers were demarcated by P27^KIP as in F. (I) The percentage of ciliated GCs in the IGL is plotted for BrdU labeled and unlabeled cells 48 h after injection. P values: 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****).