Fig. 3. Gli3 regulates proper establishment of the isthmus and cerebellum. (A-E) RNA in situ hybridization for Math1 and DAPI staining show that the external granule cell layer (EGL) is absent from the most posterior and anterior parts (arrowheads) of the isthmus-cerebellar like (Is/Cb) region in Gli3^-/- mutants, but is comparable to WT in En1-Gli3 cko mutants (arrowheads). (F-N) Immunohistochemistry on adjacent sections. (F-K) In the WT, Purkinje cells (PC) positive for calbindin and ROR (green) are organized in a several-cell-deep layer (PCL) underlying the EGL and project into the deeper Cb (arrowheads). In Gli3^-/- mutants, only a rudimentary PCL forms with disorganized projections (arrowheads), and many PC remain in clusters in the deeper Is/Cb (outlined). (H,K) In En1-Gli3 cko mutants, most PC are located within the PCL, with only some scattered PC in the underlying areas (H',K', arrowheads) and in ectopic clusters in the anterior Is (outlined). Some PC axons project into the Is (H, arrowhead). (L,M) Pax2 (green) is expressed in a scattered pattern throughout the Is and Cb (except the EGL and PCL) in WT and En1-Gli3 cko mutants, but is not expressed in the anterior (EGL-free) region in Gli3^-/- mutants and is excluded from the PC clusters (M, arrowheads). (O-Q) H+E staining of P2 sagittal sections shows the abnormal foliation pattern in En1-Gli3 cko mutants. Brain regions are outlined where necessary. Note that some of the pictures presented are composites of two images (C,D,F,G,I,J). Scale bars: 200 µm in A-N; 20 µm in H',K'; 500 µm in P-Q.