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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.
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(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.
