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First published online 29 March 2006
doi: 10.1242/dev.02351

Development 133, 1811-1821 (2006)
Published by The Company of Biologists 2006
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The level of sonic hedgehog signaling regulates the complexity of cerebellar foliation

JoMichelle D. Corrales1,2,*, Sandra Blaess1,2, Eamonn M. Mahoney1 and Alexandra L. Joyner1,2,3,{dagger}

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.


Figure 1
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  		Fig. 1. Generation of a conditional allele and verifying the generation of a null allele. Schematic representation of the gene targeting strategy (A). A neo cassette flanked with Frt sites (red triangles) and two loxP sites (yellow triangles) were inserted into the Gli2 locus using homologous recombination. ES cell clones were screened using Southern hybridization of Asp718-digested DNA with a 3' external probe (B). PCR primers floxC and floxD flanking the 3' loxP site (blue arrows, A) were used for genotyping (C). Primers floxA and floxD (blue arrows, A) were used to detect the recombined allele (C). The cerebella from Gli2{Delta}//zfd (D) and Gli2{Delta}/{Delta} (E) embryos resembled the Gli2-null cerebellum, which lacks foliation. Gli2-En1 cko mutants (G) at E16.5 appear similar in size and morphology to wild type (F). By E18.5, a reduction in foliation is observed in mutants (I) when compared with wild type (H).

 

Figure 2
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  		Fig. 2. Whole-mount and histological analysis shows decreased complexity of foliation in adult Gli2-En1 cko mutants. Cerebellar size is severely compromised along the anteroposterior (AP) axis in mutants, but the mediolateral extent is similar to wild type (A,D). Sagittal sections show fewer fissures in the vermis of the mutant (B,E). Some fissures in the mutant are barely detectable (arrows, E) and others are completely absent (asterisks, B). Horizontal sections highlight the similar mediolateral size of wild types and mutants; the mutant cerebellum is reduced in size along the AP axis (C,F, shown schematically in C',F'). The fissures in the mutant are much shallower than in wild type. Abbreviations: LS, lobulus simplex; CI, crus I; CII, crus II; PM, paramedial lobule.

 

Figure 3
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  		Fig. 3. Developmental series analysis reveals a delay and premature termination of foliation. At P0, the wild-type (A) and Gli2-En1 cko mutant (B) cerebella appear similar in size in whole mount. By P2, a slight difference in size is observed between wild type (E) and mutant (F). The mutant cerebellum at P5 (J) is clearly smaller than that of the wild-type (I). Histological analysis of sagittal sections at P0, P2, and P5 of wild-type (C,G,K) and Gli2-En1 cko (D,H,L) cerebella show shallower fissures, and a thinner EGL in mutants. (M-T) The mutant cerebellum does not increase in size after P8 (N,P,R,T) compared with the continued growth observed after P8 in wild-type (M,O,Q,S). The overall anteroposterior pattern of foliation appears intact, although the number of fissures present in P16 mutants (T) is comparable to the number present in wild type at P2 (S). (U-Z) At P0, the mutant EGL (X) is thinner than in wild type (U). By P5, the wild-type EGL has thickened to more than 10 cell layers (V), whereas the mutant EGL contains 2-4 cell layers (Y). By P8, the wild-type EGL remains thick (W), in contrast to the mutant EGL, which has been depleted (Z). Arrows indicate principle fissures forming cardinal lobes, asterisks indicate fissures forming lobules and open circles represent fissures forming sublobules. Broken yellow line indicates pial surface between two lobules; broken blue line outlines the cerebellum. Scale bar: 250 µm in C,D,G,H; 350 µm in K,L; 500 µm in O,P,S,T.

 

Figure 4
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  		Fig. 4. Laminar organization is grossly normal in Gli2-En1 cko cerebella. The internal granule layer (IGL) containing differentiated granule cells marked by NeuN is extremely sparse in mutants (D) compared with wild type (A). The PCs, marked by calbindin, are multilayered with less elaborate dendritic arborization in mutants (E), in contrast to the monolayer and complex arborization in wild type (B). The Bergmann glia marked by BLBP in green, are interspersed with the PCs (red); however, the glial fibers appear disorganized in mutants (F) compared with wild types (C).

 

Figure 5
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  		Fig. 5. Removal of Gli activator further reduces foliation in Gli2-En1 cko cerebella. Histological analysis of sagittal sections reveal that only principle fissures form in the absence of Gli1 and Gli2 (A,B). Marking differentiated neurons with NeuN shows few GCs and a thin IGL in Gli2-En1 cko;Gli1-/- mutants (C). In Smo-Nes cko mutants, only the principle fissures form in medial sections (D) and little foliation occurs in lateral sections (E). This phenotype varies (D, inset). Gli2-Nes cko mutants develop more fissures (arrow, F) than Gli2-En1 cko mutants (see Fig. 4). Scale bar: 500 µm.

 

Figure 6
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  		Fig. 6. Cell cycle genes Mycn and cyclin D1 (Ccnd1) are downregulated in Smo-Nes cko and Gli2-En1 cko cerebella. RNA in situ hybridization shows downregulation of the transcriptional target of Shh, Gli1, in Smo-Nes cko mutants at E18.5 (A), although expression of Math1 (B) indicates the presence of GCPs. Another transcriptional target of Shh, Mycn, is downregulated in Smo-Nes cko at E18.5 and Gli2-En1 cko mutants at P0, respectively (E,G), compared with wild types (C). Ccnd1, which is downstream of Mycn, is also downregulated but still present in mutants (F,H) compared with wild types (D).

 

Figure 7
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  		Fig. 7. The level of Shh signaling correlates with size and number of lobules. Histological analysis of adult cerebella shows Shh-P1 transgenics (B) have a larger cerebellum than normal (A), with longer lobules and bumps in specific locations (e.g. arrow). Shh-P1;Ptch1+/- mutants (C) also have longer lobules and an extra sublobule forms (arrow) which corresponds to a sublobule (arrow) present in the adult rat (D). Scale bar: 500 µm.

 

Figure 8
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  		Fig. 8. A graded series of Shh signaling levels generates varying degrees of fissure formation in the cerebellum. (A-L) Schematics of sagittal sections show the morphology of normal cerebella at different stages as well as adult mutants. (B) Principle fissures at E18.5 are marked with asterisks. (G) In the absence of Smo function, no fissures form. (H) Mosaic removal of Smo allows partial rescue by the remaining wild-type cells, and the principle fissures form, similar to the pattern present in E18.5 wild-type cerebella. A similar pattern of fissures forms when only Gli3 is present. (I) In the absence of Gli2, lobulation progresses slightly further, such that two additional fissures form (arrows), as in P2 wild-type cerebella. (J) Mosaic removal of Gli2 allows an additional fissure to form (arrow). (L) When excess Shh signaling occurs, lobulation progresses further than normal. Pink indicates the PC layer, and blue represents GCPs and GCs. Abbreviations: ABL, anterobasal lobe; ADL, anterodorsal lobe; CEL, central lobe; POL, posterior lobe; INL, inferior lobe; pct, precentral; pcu, preculminate; icl, intraculminate; pr, primary; dcl, declival; intercrural, itc; ppy, prepyramidal; se, secondary; uvu, uvular; po, posterolateral). Figures not drawn to scale. Scale bar: 400 µm in A-C,G-J; 425 µm in D,D; 500 µm in F,K-L.

 

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© The Company of Biologists Ltd 2006