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First published online 20 October 2004
doi: 10.1242/dev.01438

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Spatial pattern of sonic hedgehog signaling through Gli genes during cerebellum development

JoMichelle D. Corrales^1,2, Gina L. Rocco¹, Sandra Blaess^1,2, Qiuxia Guo^1,* and Alexandra L. Joyner^1,2,3,

¹ Howard Hughes Medical Institute and Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, 540 First Avenue, New York, NY 10016, USA
² Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA
³ Department of Physiology and Neuroscience, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA

View larger version (100K): [in a new window]   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.

View larger version (93K): [in a new window]   Fig. 2. Gli and Gli2 are expressed in specific cell types in the cerebellum. Expression of Gli and Gli2 co-localize with BLBP, a marker for Bergmann glia (A,D). Purkinje cells, marked with Calbindin, do not express Gli and a few express Gli2 (B,E). Gli expression is restricted to the proliferative GCPs. The inner EGL and IGL, marked by NeuN, do not express Gli at high levels (C). However, Gli2 expression is observed throughout the EGL and IGL (F). Bars indicate layers containing Bergmann glia nuclei (BG) in (A,D), Purkinje cell layer (PCL) in (B,E) and IGL (C,F). Scale bar: 50 µm.

View larger version (35K): [in a new window]   Fig. 8. Lowering Shh but not Gli levels partially rescues the Shh-P1 phenotype. Sagittal sections were analyzed for morphology. Removal of the downstream activator Gli does not affect cerebellar size (A) and does not show rescue of the mutant phenotype in double mutants (C), demonstrating that Gli is not the major activator of Shh signaling in the Shh-P1 cerebellum. When one allele of endogenous Shh was removed, a partial rescue of the Shh-P1 phenotype was observed (D). Although the cerebellum of Shh-P1; Shh+/– was larger than the Gli–/– (A), the IGL was not as thick or as irregular as the Shh-P1 IGL (B). Anterior is to the left. Scale bar: 320 µm.

View larger version (87K): [in a new window]   Fig. 3. Gli2 is required for expansion of the EGL and foliation at birth. Whole-mount analysis of WT and Gli2–/– brains reveals a cerebellar phenotype. The mutant cerebellum (D) is smaller than that of the WT (A). Cresyl Violet staining of sagittal sections through medial WT (B) and mutant (E) brains shows reduced foliation in the mutant. Math1 expression indicates the presence of GCPs in the EGL of WT (C) and mutant (F) cerebella. High magnification image of PCNA labeling in the EGL demonstrates that the proliferative layer is thinner in mutants (H) compared with WT (G). Hematoxylin and eosin staining of Gli3–/– brains shows that the EGL thickness is similar to WT (I). Region depicted in G,H is indicated in B,E. Anterior is to the left. EGL cell counts from three regions in WT and Gli2 mutant cerebellar sections were compared (J). In the mutant, regions I and III contain significantly fewer GCPs than WT at E18.5. Error bars indicate the s.d. Student's t-test was performed and showed a significant difference between WT and mutant in regions I and III (*P<0.0001). Scale bar: 200 µm in B,C,E,F; 80 µm in G,H,I.

View larger version (70K): [in a new window]   Fig. 4. The Gli2–/– phenotype is specific to the EGL. Antibody marker analysis shows that Purkinje cells marked with Calbindin (A,E) and Bergmann glia marked with BLBP (B,F) are present and their general cellular organization appears normal in Gli2–/– embryos at E18.5. Gli-lacZ is not detectable in Gli2–/– (compare C and G). However, Gli mRNA is detectable by RNA in-situ hybridization in Gli2–/– embryos (H), but its levels are much weaker than in WT (D). Anterior is to the left. Scale bar: 100 µm.

View larger version (62K): [in a new window]   Fig. 5. Shh-P1 mutants have a larger but well patterned cerebellum. Dorsal views of adult mutant brains (E) show the transgenic cerebellum is much larger than the WT (A). Cresyl Violet staining of paraffin sections of adult mutant cerebella shows enlargement of the cerebellum and a thicker IGL (indicated by bar) in medial (F) and lateral (G) sections, compared with WT (B,C). In the vermis, the phenotype is more severe in lobes III, IV, IV and IX and around the primary fissure. At P0 (inset, D,H) and P5, Gli-lacZ expression is maintained in its normal pattern in Shh-P1 mutants (H), although at higher levels than in WT (D). In sections, anterior is to the left. Scale bar: 400 µm in B,C,F,G; 250 µm in D,H.

View larger version (65K): [in a new window]   Fig. 6. The cerebellum in Shh-P1 mice overgrows after P5. Midsagittal sections of WT and mutant brains at P5 appear morphologically similar (A,E). By P8, the mutant IGL (indicated by bars) begins to appear thicker (B,F). At P14, the mutant IGL is noticeably thicker (G) compared with WT (C). The phenotype is most apparent at P28 when the IGL is also irregularly shaped (arrowheads in H), particularly around the primary fissure (arrows). [Note: Fig. 6D,H are duplicated from Fig. 5B,F and placed here for comparison to other stages.] Scale bar: 350 µm in A,E; 500 µm in B,C,F,G; 400 µm in D,H.

View larger version (76K): [in a new window]   Fig. 7. The Shh-P1 EGL is not thicker at early stages, but persists longer than in WT cerebella. Antibody staining for PCNA at P5, a marker for proliferating cells, appears similar in WT (A) and mutant (E) EGL. Cresyl Violet staining of P14 sections shows the EGL is one cell layer thick in WT (B), and three to four cell layers thick in mutants (F). PCNA labeling shows the presence of proliferating cells in the EGL at P14 in both WT and mutant (C,G). By P16, the EGL has been depleted in WT (D), but one cell layer is still present in the mutant (H). Red dashed line indicates division between two lobes. Scale bar: 50 µm.

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