Otx2 promotes granule cell precursor proliferation and Shh-dependent medulloblastoma maintenance in vivo

a Expression of Otx2 in the posterior murine cerebellum at P1, P3, P5 and P7. Otx2 protein was detected by immunofluorescence staining (red) on cerebellar sagittal sections. Nuclei were counterstained with Hoechst (blue). Last picture on the right is an enlargement of lobule X at P7. b Detection of Otx2 and proliferating cells in lobule X at P5. Mice were injected with EdU 1？h before sacrifice. Ki67 (green) and Otx2 (red) were detected by immunostaining, while EdU (yellow) was revealed with a fluorescent azide. Nuclei were counterstained with Hoechst (blue). EGL can be subdivided into two parts (white dashed line): an outer proliferative zone and an inner non-proliferative zone. c Co-expression of Otx2 and GFP in the cerebellum of Otx2+/Otx2-GFP transgenic mice at P7. Otx2 (red) and GFP (green) were detected by immunostaining, while nuclei were counterstained with Hoechst (blue). Merge of the two images shows a perfect overlap between Otx2 and GFP expression. d FACS analysis of GCPs purified from P7 Otx2+/Otx2-GFP cerebella. GFP, Ki67 and EdU incorporation were detected by fluorescence staining on dissociated GCPs and analysed by FACS. Two populations of GCPs (Otx2+ and Otx2？) can be identified based on the expression of GFP. e Quantification of Ki67 expression and EdU incorporation in these two populations at P3, P5 and P7 indicate that Otx2+ GCPs have a higher proliferative potential than Otx2？ GCPs. SSC side scatter, P postnatal day. Scale bar: 200？μm (a, c) and 50？μm (b). Data are represented as mean？±？sem. *p？<？0.05, **p？<？0.00