Fig. 5. Expression analysis of Smads in the developing cerebellum. In situ hybridisation was performed on vibratome sections of chick cerebellae with probes to Smad1, Smad5 and Smad8. (A) Hybridisation with a Smad1 probe at HH38 reveals strong expression in the EGL. (B,C) At HH42 Smad1 expression remains restricted to the EGL. (D) Double labelling with Smad1 and the mitosis marker Phospho-Histone3 (brown nuclei) reveals Smad1 expression (blue staining) localised in the highly proliferative external EGL. (E) Hybridisation with a Smad5 probe at HH38 shows expression in the EGL and the IGL. (F,G) At HH42, Smad5 expression is weak in the EGL but strong in the IGL, particularly in cells just below the Purkinje cell layer. (H) Double labelling with Smad5 and the Purkinje cell marker calbindin (brown neurones) reveals Smad5 labelling (blue staining) in granule neurons just below the Pc layer. (I,J) Hybridisation with a Smad8 probe at HH38 (I) and HH42 (J) shows no expression in any layer of the developing cerebellum. (K,L) Expression of Smad8 in cells of a motor nucleus at the ventral medulla. (M-O) Vibratome sections of mouse P4 (M) and chick HH42 (N,O) cerebellae immunostained with the phospho-specific Smad 1/5/8 antibody show the presence of phosphorylated forms of Smad proteins mainly at the IGL, together with few nuclei leaving the iEGL (arrows), which should correspond with Smad5 mRNA expression. The location of Purkinje cell bodies is indicated by broken lines.