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Fig. 1. Shh and the downstream factor genes Gli, Gli2 and Gli3 are expressed in the developing cerebellum. RNA in situ hybridization shows expression of Shh in the PCL at E18.5 (A), P5 (E) and adult (P28) (I). Shh expression is strongest in anterior regions during early stages (arrows), as well as posterior to the secondary fissure and appears homogenous in the PCL in the adult. Inset in I is a high magnification image of Purkinje cell layer indicated by box. ß-Gal activity from the Gli locus reveals positive Shh signaling in areas corresponding to Shh expression. At E18.5, Gli-lacZ is strongest anteriorly (arrow) and expression is also observed posterior to the secondary fissure (B). In lateral sections, strong Gli-lacZ is observed only in the anterior cerebellum (inset, B). By P5, the intermediate region expresses Gli-lacZ and expression remains stronger in the EGL and PCL anteriorly and posterior to the secondary fissure (F). In the adult, PCL expression is homogenous although IGL expression was higher after 24 hours incubation anterior to VIa and posterior to the secondary fissure (J). Gli2-lacZ was expressed in the EGL and deeper layers along the AP axis at E18.5 (C) and P5 (G). Staining appears weaker between anterior and posterior regions due to a thinner EGL at E18.5. Gli2-lacZ in the adult is present in the IGL and PCL equally along the AP axis (K). At E18.5, Gli3 expression is detected uniformly in the EGL and deeper layers (D). By P5, Gli3 remains homogeneous along the AP axis, and stronger expression is observed in the outer EGL (H, and inset). In the adult, Gli3 expression remains broad. Anterior is to the left. Scale bar: 125 µm in A,B,C,D; 250 µm in E,F,G,H; 500 µm in I,J,K,L.

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