Fig. 1. Distinct temporal roles of Gli3 in regulating midbrain and cerebellum development in mouse. (A-H) Hematoxylin and Eosin (H+E) staining of midline (A-D) and lateral (E-H) E18.5 sagittal brain sections. (B,F) In Gli3^-/- mutants, the dorsal midbrain is enlarged and the distinct morphology of the inferior colliculus (Ic) and superior colliculus (Sc) is lost. Similarly, the isthmus (Is) and cerebellum (Cb) are not clearly separated and contain cell clusters (red arrowheads). The Cb is not foliated. The morphology of the ventral mid/hindbrain (vMh) appears normal. (C,G) In En1-Gli3 cko mutants, the Sc, Ic, Is and Cb (arrow) are enlarged, and tectum, Is and Cb are morphologically distinct from one another. The Cb foliation pattern is abnormal. (D,H) In Nes-Gli3 cko mutants, the Sc, Ic, Is and Cb are morphologically distinct, but the Sc, Ic and Is are increased in size. (I-K) Immunohistochemistry for tyrosine hydroxylase (TH) shows no change in dopaminergic neurons (green, arrows) in the mutants. DAPI staining is in blue. (L) Quantitative assessment of Cb and Sc size in wild-type (WT) and Nes-Gli3 cko brains as means of samples from three different animals ±s.e.m. Student's t-test was performed. (M-O) H+E staining of E10.5 sagittal embryo sections. Note the increased size of the ventricle and increased thickness and abnormal morphology of the Is/r1 region in Gli3^-/- and En1-Gli3 cko mutants. (P) On the left is a schematic of Shh and Gli expression in the ventral (V) and dorsal (D) embryonic mes/r1. On the right, Shh signaling in the ventral and dorsal mes/r1: high levels of Shh induce Gli activator (GliA2/3, green) and inhibit (red) the formation of Gli3 repressor (Gli3R, purple) ventrally. Low levels of Shh decrease the formation of Gli3R dorsally. Gradients indicate high to low levels of expression/signaling. Scale bars: 500 µm in A-H; 250 µm in I-K,M-O.