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

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The level of sonic hedgehog signaling regulates the complexity of cerebellar foliation

JoMichelle D. Corrales^1,2,*, Sandra Blaess^1,2, Eamonn M. Mahoney¹ 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.

Author for correspondence (e-mail: joyner{at}saturn.med.nyu.edu)

Accepted 8 March 2006

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Foliation of the mouse cerebellum occurs primarily during the first 2 weeks after birth and is accompanied by tremendous proliferation of granule cell precursors (GCPs). We have previously shown that sonic hedgehog (Shh) signaling correlates spatially and temporally with fissure formation, and that Gli2 is the main activator driving Shh induced proliferation of embryonic GCPs. Here, we have tested whether the level of Shh signaling regulates the extent of cerebellar foliation. By progressively lowering signaling by removing Gli1 and Gli2 or the Shh receptor smoothened, we found the extent of foliation is gradually reduced, and that this correlates with a decrease in the duration of GCP proliferation. Importantly, the pattern of the remaining fissures in the mutants corresponds to the first fissures that form during normal development. In a complementary manner, an increase in the level and length of Shh signaling results in formation of an extra fissure in a position conserved in rat. The complexity of cerebellar foliation varies greatly between vertebrate species. Our studies have uncovered a mechanism by which the level and length of Shh signaling could be integral to determining the distinct number of fissures in each species.

Key words: Gli1, Gli2, Gli3, Shh, Cerebellum, Patterning, Mouse

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The relatively few well-defined cell types within the cerebellum and the stereotypical foliation pattern make the cerebellum particularly amenable to the study of morphogenesis in the central nervous system (CNS). The mammalian cerebellum consists of a central vermis and two lateral hemispheres, each with its own sets of fissures. The complexity of the foliation pattern varies between species of mammals, depending on the proprioceptive input to the cerebellum. For example, the cerebellar vermis of many inbred mouse strains consists of eight major lobules with few sublobules, whereas the rat vermis consists of ten lobules and contains more sublobules. Furthermore, some inbred strains of mice have one or two additional partial lobules corresponding to additional lobules in rat. Although the basic ten lobules present in the rat are conserved in human, each human cerebellar lobule is extensively subdivided into many sublobules. The conservation of morphology within and across species suggests that patterning of cerebellar folia is genetically regulated. An important question is what factors determine the degree of foliation in different species.

Foliation of the rodent cerebellum is generated in distinct phases (Altman and Bayer, 1997). First, the smooth cerebellar surface is divided into five cardinal lobes by four principle fissures in the vermis (see Fig. 8). Next, the process of lobulation divides the cardinal lobes into the individual lobules present in the adult cerebellum. Some of the lobules are then further subdivided into sublobules, and finally each lobule grows to a specific size. In the mouse and rat, the emergence of the five cardinal lobes is observed by birth. The foliation pattern elaborates for 2 weeks after birth in mouse and 3 weeks in rat. Although a number of theories have been proposed (Lauder et al., 1974; Mares and Lodin, 1970), it remains unknown how the conserved position of the fissures is determined, and what genetic mechanisms regulate the size and complexity of the lobes. It is also not known whether positioning of the folia and regulation of the number of fissures are independent or inter-related events.

The cerebellum undergoes over a 1000-fold increase in volume during development (Goldowitz et al., 1997). Postnatally, this is largely due to the proliferation of the GCPs and occurs simultaneously with the formation and growth of the folia. The generation of granule cells and the process of cerebellar growth and foliation appear to be related, as mice or rats in which the granule cell population is compromised develop smaller cerebella with reduced foliation (Aruga et al., 1998; Doughty et al., 1998; Dussault et al., 1998). GCP proliferation is dependent on interactions with Purkinje cells (PCs), as demonstrated by the observed decrease in granule cell number when PCs are lost because of cell ablation or genetic mutation (Caddy and Biscoe, 1979; Herrup, 1983; Sidman et al., 1962; Smeyne et al., 1995; Wetts and Herrup, 1982).

A number of lines of evidence suggest the mitogenic effect of PCs on granule cells is mediated by sonic hedgehog (Shh), a secreted factor expressed in PCs from E17.5 onwards in the mouse. First, Shh increases and sustains proliferation of GCPs in culture (Dahmane and Ruiz-i-Altaba, 1999; Lewis et al., 2004; Wallace, 1999; Wechsler-Reya and Scott, 1999). Second, Shh expression at E17.5 to early postnatal stages is spatially restricted to the regions where fissures form first (Corrales et al., 2004; Lewis et al., 2004). Third, once the cells become postmitotic, the response of GCPs to Shh is greatly reduced (Corrales et al., 2004). Fourth, progressive deletion of Shh in mouse PCs using an L7-Cre transgene (Lewis et al., 2004) or inhibition of Shh by antibodies in the chick cerebellum (Dahmane and Ruiz-i-Altaba, 1999) results in reduced GCP proliferation and reduced foliation. Inactivation of mouse Shh during neural tube closure using Pax2-Cre results in a more severe phenotype (Lewis et al., 2004); however, we have recently shown an early requirement for Shh in setting up a normal cerebellar primordium (Blaess et al, 2006). Thus, it remains unclear whether Shh is required after E16.5 for any fissures to form and whether Shh plays an instructive role in determining the pattern of folia.

All Shh signaling in patterning the mouse spinal cord is mediated by the three Gli transcription factors (Bai, 2004; Wijgerde et al., 2002). Genetic studies in the mouse have demonstrated that Gli3 functions primarily as a repressor, whereas Gli1 and Gli2 function as activators in the spinal cord (reviewed by Jacob and Briscoe, 2003). We have recently demonstrated that Gli2 is the major activator downstream of Shh in embryonic GCPs after E16.5 (Corrales et al., 2004). Although GCPs are specified normally in Gli2-null mutants, the external granule layer (EGL) is thinner and foliation is not initiated at E18.5. However, the lack of fissure formation could reflect either a delay in foliation because of reduced proliferation, or an absolute halt in foliation.

In a previous study, we identified a direct correlation between the spatial and temporal onset of fissure formation and elevated levels of Shh signaling in the developing cerebellum (Corrales et al., 2004). Here, we have tested the hypothesis that the level of Shh signaling determines the complexity of foliation in different mammals by analyzing mouse conditional mutants in which Shh signaling was progressively reduced or raised. Strikingly, we found that foliation proceeds in the absence of Gli2, but the progression of lobulation and growth of cerebellar lobules is delayed and prematurely halted. When Gli1 is removed in addition to Gli2, foliation is further reduced. When all Shh signaling is removed after the cerebellar primordium is formed, all cerebellar foliation is inhibited because of a rapid depletion of GCPs after E17.5. Finally, raising Shh levels in PCs leads to formation of an additional fissure with a position conserved in rat. Thus, the level of Shh signaling regulates the extent of cerebellar foliation, but not the pattern.

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Generation of a targeting construct
A 3.0 kb NotI-SpeI genomic fragment of Gli2 containing intron 6 was inserted into a targeting vector upstream of a neo cassette flanked with Frt sites, with one loxP site 3' to the neo cassette and a TK cassette. Another loxP site containing an Asp718 site was inserted into a SpeI site within intron 9 in a 9.6 kb NotI-HindIII genomic fragment of Gli2. This fragment was then inserted 3' to the loxP site contained in the original vector (Fig. 1A).

Breeding and genotyping of mice
One targeted W4 (Auerbach et al., 2000) ES cell clone was identified (Matise et al., 2000) from 122 G418 and GANC resistant cell clones by Southern blot analysis using Asp718 and a 3' external probe from nucleotides 1290-2092 of the Gli2 cDNA (encompassing exons 10-12) or using HindIII and an internal probe containing neo (see Fig. 1B). ES cell chimeras were generated through injection of C57BL/6 blastocysts (Skirball Transgenic Facility). Heterozygous Gli2^floxneo/+ offspring were bred with ACTB-Flpe mice (Rodriguez et al., 2000) to produce Gli2^flox/+ heterozygotes that were outbred to Swiss Webster mice.

We verified that recombination between the loxP sites produces a null allele by generating a deleted allele (Gli2) and intercrossing them or breeding with mice carrying a null allele (Gli2^zfd/+) (Mo et al., 1997). Both Gli2^/ and Gli2^/zfd E18.5 embryos displayed the same cerebellar phenotype described in Gli2^zfd/zfd embryos (Corrales et al., 2004) (Fig. 1D-E; data not shown). Other phenotypes observed in Gli2^zfd/zfd embryos (Matise et al., 1998; Mo et al., 1997; Palma and Ruiz i Altaba, 2004), such as lethality at birth, hydrocephaly, a curved body axis and abnormal patterning of the E10.5 ventral spinal cord were also present in Gli2^/ and Gli2^/zfd mutants (C. B. Bai, unpublished; data not shown).

Double heterozygous mice (En1^Cre/+;Gli2^zfd/+) were crossed to Swiss Webster females to produce En1^Cre Gli2^zfd/En1⁺ Gli2⁺ mice in which intrachromosomal recombination linked the En1^Cre (Kimmel et al., 2000) and Gli2^zfd (Mo et al., 1997) alleles. These mice were crossed to Gli2^flox/flox mice to generate the conditional mutants En1^Cre Gli2^zfd/En1⁺ Gli2^flox (Gli2-En1 cko). En1^Cre/Gli2^zfd mice which were also heterozygous for a null lacZ knock-in allele of Gli1 (Bai et al., 2002), were bred with Gli2^flox/+ mice carrying the Gli1^lacZ allele to generate En1^Cre/Gli2-cko;Gli1^lz/lz mice. Other mutant alleles were genotyped as previously described: Nestin-Cre (Tronche et al., 1999), Smo^flox and Smo^rec (Long et al., 2001) and Shh-P1 (Riccomagno et al., 2002). All mouse lines were maintained on an outbred Swiss Webster background. Although the cerebellar foliation pattern varies slightly between strains of mice (Inouye and Oda, 1980), in this outbred background we observed consistent foliation patterns in wild-type mice. The uvular sulcus (see Fig. 8 for definition), which is absent in some strains, was always present. The intraculminate fissure within lobule V, which varies in length between strains, was shallow in 80% of mice and the precentral a fissure that produces an additional tiny anterior lobule in FVBN mice was rarely present.

Noon of the day a vaginal plug was designated as E0.5. The day of birth was designated as P0. Adults were designated as P28 or older. For genotyping the Gli2^flox allele, primers flanking the 3' loxP site were used: floxC, 5'-AGGTCCTCTTATTGTCAGGC-3'; floxD, 5'-GAGACTCCAAGGTACTTAGC-3' (Fig. 1A). The mutant allele produced a 247 bp product, and the wild-type allele a 231 bp band (Fig. 1C). For detecting the recombined allele, Gli2^/+, primer sequences were 5'-CTTATGGACATCTGTCTGCC-3' (floxA) and 5'-GAGACTCCAAGGTACTTAGC-3' (floxD, see above). The recombined allele produces a 500 bp band (Fig. 1C).

Histology, immunocytochemistry and RNA in situ hybridization
For lacZ staining, brains were dissected fresh, embedded in OCT (Tissue-Tek) and sectioned at 20 µm. For histology and mice older than P8, either intracardiac perfusion was performed followed by immersion fix of tissue in paraformaldehyde (PFA) at 4°C, or freshly dissected brains were fixed overnight at 4°C using Carnoy. Tissue was then processed for paraffin embedding and sectioned at 7 µm. For consistency, sections analyzed from the vermis were limited to the most medial 100 µm. TUNEL assay was performed using ApopTag (Chemicon). Antibody staining was performed according to standard protocol. The following primary antibodies were used: rabbit -BLBP (gift from N. Heintz), 1:3000; mouse -calbindin (Sigma), 1:4000; rabbit -cleaved caspase 3 (Cell Signaling), 1:2000; rabbit -GFAP (Chemicon), 1:2000; mouse IgG -NeuN (Chemicon), 1:2000. RNA in situ hybridization on sections was performed using standard methods. Detailed protocols are available at http://saturn.med.nyu.edu/research/dg/joynerlab

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Generation of Gli2^flox conditional mutant mice
In order to reduce Shh signaling and determine the requirement for positive Shh signaling through Gli2 in cerebellar foliation, we generated mice carrying a conditional Gli2 mutant allele, Gli2^flox (Fig. 1A-C; see Materials and methods). Two loxP sites were inserted flanking exons 7 and 8, which are upstream of the exons encoding the zinc fingers. Deletion of these exons results in a frameshift mutation following splicing of mRNA from exon 6 to 9. The floxed allele was found to have wild-type activity, as Gli2^flox/flox and Gli2^flox/zfd mice appear normal (data not shown) and the deleted allele confers a null phenotype (Fig. 1D,E; see Materials and methods).

Deletion of Gli2 in the midbrain and cerebellum after E9 rescues the lethal phenotype of Gli2 null mutants
In order to generate a viable mutant in which Gli2 was deleted from all cerebellar precursors, we used the En1^Cre/+ mouse line (Kimmel et al., 2000) in which Cre deletes target genes by E9.0 (Li et al., 2002) in all cells of the mesencephalon and rhombomere 1 (which gives rise to the cerebellum dorsally) (Zervas et al., 2004). En1 and Gli2 are separated by only 1.1 cM on chromosome 1; thus, mice in which En1^Cre and Gli2^zfd are linked were crossed to Gli2^flox/flox mice to generate conditional mutants, referred to as Gli2-En1 cko (conditional knockout) mutants (see Materials and methods).

View larger version (35K): [in this window] [in a new window]   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//zfd (D) and Gli2/ (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).

Gli2 is required for the full extent of growth and elaboration of the cerebellar lobes, but not patterning
We next determined whether cerebellar foliation could occur after birth when positive Shh signaling is greatly reduced. Whole-mount analysis of Gli2-En1 cko adult brains demonstrated an extreme decrease in the size of the cerebellum compared with wild type, whereas the rest of the brain appeared normal in size (Fig. 2A,D; data not shown). Of significance, fissures were observed in adult mutant cerebella, demonstrating that some foliation can occur in the absence of Gli2. The mediolateral extent of the adult mutant cerebellum appeared to be decreased less than the anteroposterior axis (arrows, Fig. 2A,D).

Horizontal and sagittal sections confirmed that the length of the Gli2-En1 cko cerebellum along the mediolateral axis was not as drastically reduced compared with the anteroposterior axis and that the basic laminar structure was intact (Fig. 2C,F, shown schematically in Fig. 2C',F'). Strikingly, the cardinal lobes were present in the mutant vermis and hemispheres, although the number of additional lobules was greatly reduced compared with wild type. In the vermis of Gli2-En1 cko mutants, the fissure that normally divides the anterobasal lobe was absent or extremely shallow in six out of seven mutants, and the central lobe was divided by a shallow fissure in all mutants (arrows, Fig. 2B,E). In the hemispheres, the paramedial lobule was not subdivided in mutants and the fissures subdividing crus I and crus II were shallower in mutants than in wild types (Fig. 2C,F). Thus, the foliation pattern seen in Gli2-En1 cko adults seems to reflect formation of only the earliest forming fissures, suggesting that foliation is initiated, but then does not progress to completion.

Progression of foliation is delayed and terminated prematurely in Gli2-En1 cko mutants
We next determined whether the sequential generation of folia progressed normally but did not reach completion in Gli2-En1 cko mutant cerebella. Although P0 wild-type and mutant cerebella appeared similar in whole mount (Fig. 3A,B), by P2, Gli2-En1 cko mutant cerebella were obviously smaller than normal along the AP axis (Fig. 3E,F), and by P5 the difference was striking (Fig. 3I,J). In wild-type brains, the cerebellum continues to grow until P16, whereas in the Gli2-En1 cko mutants, the cerebellum did not appear to increase in size after P8 (Fig. 3M,N,Q,R).

View larger version (54K): [in this window] [in a new window]   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.

The EGL is depleted earlier than normal in Gli2-En1 cko mutants
We next determined the cellular basis of the decreased foliation and growth in Gli2-En1 cko cerebella. As Gli2-null mutants have a thinner EGL at E18.5 (Corrales et al., 2004) and GCP proliferation is required for foliation, we examined the EGL in postnatal Gli2-En1 cko mutants. At birth, the wild-type EGL is thicker at the base of each fissure and overall is thinnest in the central region; however, this variation is minimal at later stages. We found that in the wild type, the EGL varied from two to four cells; however, the EGL in mutants was only one or two layers thick (Fig. 3U,X). In addition, the difference in thickness was more drastic at the base of the fissures than at the crowns of the lobes. At P5, the Gli2-En1 cko mutant EGL contained one to four layers, rather than the eight to ten layers present in the wild type (Fig. 3V,Y). Strikingly, whereas the eight to ten cell layers of GCPs persisted in the wild-type EGL to P8, the mutant EGL was completely depleted of cells by P8 (Fig. 3W,Z). In wild-type mice, the EGL persisted and the cerebellum continued to grow until P16. In summary, the reduced thickness of the EGL in Gli2-En1 cko mice as early as E18.5 correlates with a delay in onset of foliation. Furthermore, the delay and a premature depletion of the EGL correlate with a reduction in the complexity of foliation.

View larger version (93K): [in this window] [in a new window]   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.

View larger version (93K): [in this window] [in a new window]   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).

A previous in vitro study suggested a role for Shh in differentiation of the Bergmann glia (Dahmane and Ruiz-i-Altaba, 1999). To determine whether Bergmann glia were altered in Gli2-En1 cko mutants, we used antibody labeling for the Bergmann glial markers BLBP and GFAP. Cells positive for both antibodies were present in Gli2-En1 cko mutants, demonstrating that Shh signaling through Gli2 is not required for Bergmann glial specification (Fig. 4C,F; data not shown). However, Bergmann glia were morphologically abnormal. Although the soma of Bergmann glia were interspersed among the PCs in Gli2-En1 cko mutants, their glial fibers were misshapen and disorganized compared with the ordered and linear morphology of wild-type Bergmann glial fibers (Fig. 4C,F). This phenotype could reflect either a direct role for Shh signaling through Gli2 in Bergmann glial maturation, or a secondary defect resulting from disorganization of the PC layer and reduction in GCs.

Removing the Gli1 activator further reduces foliation in Gli2-En1 cko cerebella
We have previously shown that in the cerebellum of E18.5 Gli2 null mutants, expression of Gli1 is drastically reduced but not absent and Gli1 mutants have a normal cerebellum (Corrales et al., 2004). To determine whether Gli1 contributes activator function in Gli2-En1 cko mutants that is responsible for the low level of GCP proliferation and foliation present in Gli2-En1 cko mutants, we generated Gli2-En1 cko mice with reduced or no Gli1. We analyzed eight litters from a cross between En1^Cre Gli2^zfd/En1⁺ Gli2⁺;Gli1^lz/+ and Gli2^flox/f+;Gli1^lz/+ mice, but only one Gli2-En1 cko;Gli1^lz/lz mouse survived, as Gli2^+/-;Gli1^-/- mice have an extremely low survival rate (Park et al., 2000). Gli2-En1 cko;Gli1^lz/+ mice, however, were generated with the expected frequency. Although Gli2-En cko;Gli1^lz/+ mice had the same phenotype as Gli2-En1 cko mice (data not shown), the Gli2-En1 cko;Gli1^lz/lz mutant cerebellum had a decreased number of fissures compared with Gli2-En1 cko mutants (Fig. 5B,C compare with Fig. 3T). In medial sections of the cerebellum, only the five cardinal lobes could be discerned. The decreased number of lobules that formed in the Gli2-En1 cko;Gli1^lz/lz mutant shows that Gli1 does indeed contribute to foliation in Gli2-En1 cko mutants.

Shh signaling is required for cerebellar foliation
Our finding that removing both Gli1 and Gli2 activators did not inhibit formation of the cardinal lobes raised the issue of whether Shh is required at all for the initial simple foliation pattern seen in all mammals. Although conditional removal of Shh using Pax2-Cre and L7-Cre did not fully inhibit foliation, the degree of recombination in these mutants is not clear (Lewis et al., 2004). We therefore analyzed cerebellar development in the absence of Smo, by generating viable conditional mutants for Smo in the brain using a floxed Smo allele (Long et al., 2001). As the cerebellar primordium is compromised in En1^Cre/+;Smo^flox/- (Smo-En1 cko) mutants by E12.5 and the mutants die at birth (Blaess et al., 2006), we used Nestin-Cre transgenic mice (Graus-Porta et al., 2001; Tronche et al., 1999) to delete Smo throughout the CNS, and in rhombomere 1 beginning around E11 (Blaess et al, 2006). Approximately 10% of Nestin-Cre;Smo-cko (Smo-Nes cko) mutants survive to three weeks [data not shown; see Machold (Machold, 2003)]. The cerebellar primordium of these mutants is grossly normal at E16.5, although slightly reduced in size (Blaess et al., 2006). The overall size of the P21 Smo-Nes cko mutant brains was smaller than wild type, although the cerebella showed a more pronounced reduction in size. As in the Gli2-cko mutants, the AP axis of the cerebellum was more dramatically reduced than the ML axis. Interestingly, histological analysis of midsagittal sections of P16-21 Smo-Nes cko brains showed a foliated cerebellum that consisted of five cardinal lobes in all but one mutant (n=4), although the four principle fissures were very shallow (Fig. 5D). In one mutant, extremely shallow fissures were observed corresponding to the positions of three of the four principle fissures (Fig. 5D, inset). Furthermore, the depth of the fissures in the medial vermis varied between animals. However, a basic layered cytoarchitecture with an IGL was present. By contrast, in some regions of lateral sections of P21 Smo-Nes cko cerebella foliation was limited to slight indentations only in the central and posterior regions (Fig. 5E).

View larger version (96K): [in this window] [in a new window]   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.

The foliation present in Gli2-En1 cko mutants was not due to inefficient recombination of Gli2^flox, as Gli1 was not detected in P2 medial or lateral cerebellar sections in these mutants (see Fig. S1C,D in the supplementary material; data not shown). We analyzed the cerebellum phenotype of Nestin-Cre;Gli2^flox/- (Gli2-Nes cko) mice as the mosaicism found using Nestin-Cre was expected to allow foliation to progress further than in Gli2-En1 cko mutants. Indeed, compared with Gli2-En1 cko mutants, the Gli2-Nes cko vermis contained a more defined intercrural fissure (arrow, Fig. 5F) and a thicker IGL than in Gli2-En1 cko mice. Recombination in these mutants was also mosaic, as demonstrated by patchy expression of Gli1 in Gli2-Nes cko mutants at P0 (see Fig. S1E,F in the supplementary material).

Shh is not absolutely required for Mycn and cyclin D1 expression
The proto-oncogene Mycn (previously Nmyc) is a potential downstream effector of GCP proliferation via Shh signaling, as it regulates proliferation and is a direct target of Shh in GCPs (Kenney et al., 2003). Mycn probably mediates GCP proliferation via cyclin D1, as cyclin D1 mRNA (Ccnd1) is also upregulated in response to Shh in GCPs (Kenney and Rowitch, 2000). We therefore examined the expression of Mycn and Ccnd1 in the absence of Gli2 in the cerebellum. In the wild-type cerebellum at P0, Mycn and Ccnd1 expression was observed in the EGL along the anteroposterior axis and some Ccnd1 expression was also detected in a deeper layer, which could correspond to the Bergmann glial layer (Fig. 6C,D). This expression pattern differs from that of Shh and Gli1, which are higher in the anterior cerebellum during early postnatal stages (Corrales et al., 2004). At P0, expression of both Mycn and Ccnd1 was reduced in the EGL of Gli2-En1 cko mutants, although expression of both genes was not abolished (Fig. 6E,F). It is likely that the reduction in expression is due in part to the decreased number of GCPs in the EGL, in addition to a direct effect of decreased Shh signaling.

One possibility was that the remaining Mycn and Ccnd1 expression was due to activator function of Gli1 or Gli3. We tested this by analyzing Smo-Nes cko mutants. In lateral sections of E18.5 Smo-Nes cko mutants, where recombination was complete, based on Gli1 expression (compare Fig. 6A and Fig. S1A in the supplementary material), both Mycn and Ccnd1 were downregulated, although some expression of both genes persisted (Fig. 6G,H). Thus, although Shh signaling through Gli activators is not absolutely required for the expression of Mycn and Ccnd1, it upregulates their expression.

Increased Shh signaling produces a more complex foliation pattern
Based on our finding that a reduction in Shh signaling results in less foliation with a corresponding reduction in the temporal length of GCP proliferation, we addressed whether increasing Shh could induce additional foliation We previously have shown that Shh-P1 transgenic mice (Shh-P1) (Riccomagno et al., 2002) have increased levels of Shh signaling and GCP proliferation extends for an additional 2 days (Corrales et al., 2004). Furthermore, these mutants develop a larger cerebellum with a thicker and irregular IGL. Upon more extensive examination of many mutants, we discovered that bulges in the IGL of Shh-P1 cerebella were consistently found in specific positions (Fig. 7B). This suggested that an underlying genetic mechanism controls where bulges form when the number of GCPs is increased. To test whether further increasing Shh signaling can lead to formation of fissures in defined positions, we generated Shh-P1 mice which were heterozygous for a mutation in patched (Ptch1), a receptor for Shh which functions to inhibit Shh signaling (Goodrich et al., 1997). Consistent with our prediction, Shh-P1;Ptch1^+/- mice not only had larger cerebella than Shh-P1 mice, but also had an extra sublobule on the rostral face of lobule VI (n=3/4) (arrow, Fig. 7C). Interestingly, the position of this sublobule is reminiscent of sublobule VI-d in the rat cerebellum (arrow, Fig. 7D). In combination with the decreased fissure formation in Gli2-cko and Smo-cko mutants, these results demonstrate a direct link between the level of Shh signaling and complexity of the cerebellar foliation pattern via regulation of GCP proliferation. The Shh-P1 transgene did not, however, enhance foliation in Gli2-En1 cko cerebella (see Fig. S2B,C in the supplementary material), demonstrating that the remaining Gli proteins are not sufficient to drive additional GCP proliferation.

View larger version (97K): [in this window] [in a new window]   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).

View larger version (34K): [in this window] [in a new window]   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.

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Despite there being a simpler foliation pattern in the Smo cko and Gli2 cko mutants, the patterns seen along the anteroposterior and mediolateral axes represent the conserved pattern seen in all mammals. This result suggests that Shh signaling does not determine the position of the fissures, or the differential patterning of the vermis and hemispheres. Shh is instead a permissive factor for foliation. Our discovery supports a previously proposed model that positioning of the fissures is determined by interactions between PCs and mossy fibers as well as GC parallel fibers (Altman and Bayer, 1997). Another theory for the mechanism of foliation is that differential rates of proliferation in the EGL (with a greater rate in the depths of the fissures) causes buckling forces to generate fissures (Mares and Lodin, 1970). Although we cannot exclude this model, as Shh signaling is even along the base and the crown of the lobules (Corrales et al., 2004), Shh does not appear to influence the complexity of foliation by regulating the rate of proliferation.

View larger version (49K): [in this window] [in a new window]   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.

Our results show that only a very minimal level of Shh signaling is required for the initial phase of cardinal lobe formation, whereas a higher level and sustained signaling is necessary for partitioning of the cardinal lobes into lobules and sublobules. Furthermore, the cerebellar phenotype of Gli2-En1 cko mice demonstrates that Shh signaling through Gli2 is not required for generation of the cardinal lobes, but instead is required to maintain the proliferative pool of GCPs such that a sufficient number of GCs are generated to achieve full lobe growth, and to complete the lobulation and sublobulation processes.

Gli2 is not required for cell type specification in the cerebellum
It was previously unknown whether Shh signaling through Gli2 regulates cell type-specific differentiation in the cerebellum. In the spinal cord, Shh signaling through Gli2 is required for the induction of the two most ventral cell types (Matise et al., 1998; Mo et al., 1997), and both Gli2 and Gli3 influence the normal differentiation of the other ventral cell types (Bai, 2004; Stamataki et al., 2005). A high level of positive Shh signaling is restricted to the proliferating GCPs and Bergmann glia (based on Gli1 expression) and thus could influence their differentiation (Corrales et al., 2004). We found that in Gli2-En1 cko mutants both of these cell types are in fact generated, although the GC population is reduced in number. The depletion of GCPs in Gli2 cko mutants probably causes a less compact IGL to form and the lack of spreading out of the PCs into a monolayer. Furthermore, the decreased number of differentiated GCs could account for the abnormal PC dendrite morphology in Gli2 cko mutants. Thus, the defects in GCs and PCs are probably not due to direct effects on differentiation of either cell type.

Although Bergmann glia form in Gli2 cko mutants, their fibers are disorganized. As Bergmann glial fibers are known to enwrap synapses on PC dendrites (Yamada et al., 2000), the Bergmann glial phenotype could be secondary to the observed abnormal PC morphology. Conversely, Bergmann glial fibers are thought to shape the dendritic morphology of PCs (Lordkipanidze and Dunaevsky, 2005); therefore, abnormal glial development may cause secondary effects on PC development. Consistent with this, Gli1 and Gli2 are both expressed in Bergmann glia (Corrales et al., 2004), and thus Shh signaling could have a direct effect on the final steps of Bergmann glia differentiation. The expression of the late marker GFAP in mutants shows that Bergmann glia differentiation nevertheless proceeds quite far. Furthermore, although disorganized, the glial fibers extend to the pial surface of the cerebellum as in wild-type mice. Finally, the Bergmann glia in Gli2 cko mutants appear similar to those in agranular rat cerebella (Doughty et al., 1998). A cell type specific conditional knock-out of Gli2 in Bergmann glia is needed to resolve which cellular defects are primary versus secondary.

Shh is not absolutely required for Mycn and Ccnd1 expression
Shh signaling has been found to regulate proliferation in the skin and hair by inducing cell cycle genes (Fan and Khavari, 1999; Mill et al., 2003; Mill et al., 2005) and both Mycn and Ccnd1 are upregulated by Shh signaling in GCPs (Kenney et al., 2003; Oliver et al., 2003). Interestingly, mice mutant for both Ccnd1 and Ccnd2 have a small cerebellum (Ciemerych et al., 2002), with a foliation pattern similar to that observed in Gli2-En1 cko mutants. We unexpectedly found that although Mycn and Ccnd1 are reduced in the absence of either Smo at E18.5 or Gli2 at P0, expression is not abolished. In addition, Mycn expression levels do not correlate spatially in the cerebellum with the levels of Shh or the downstream target Gli1. Thus, signals other than Shh must also regulate Mycn in the developing cerebellum. Consistent with the persistent expression of Mycn and Ccnd1 in our mutants, a proliferating EGL is sustained in Gli2-En1 cko mice until P8 and until birth in Smo-Nes cko mice. Furthermore, cell death is not a contributing factor to the cerebellar phenotypes in En1-Gli2 cko or Nes-Smo cko mutants, as TUNEL assay and immunostaining for activated caspase 3 did not reveal any differences in cell death between wild types and mutants at E18.5 (data not shown). Thus, Shh is clearly required to increase the level of GCP proliferation after E17.5, and to enable the process of foliation.

Shh and formation of more complex cerebella
In this study, we set out to test our prediction that the length and level of Shh signaling determines the extent of foliation in different mammals by controlling the amount of GCP proliferation in specific areas of the cerebellum (Corrales et al., 2004). The genetic mutants we constructed have a graded series of Shh signaling in the postnatal cerebellum, from a complete lack of Shh signaling through upregulated Shh signaling. The resulting cerebellar phenotypes correspondingly progress from no foliation, through immature foliation patterns, to an increased complexity of foliation. The size of the cerebellum also increases in conjunction with a greater elaboration of foliation. Furthermore, the phenotypes correlate with the length of GCP proliferation, their number and the level of Mycn transcription in GCPs. Thus, we have demonstrated that the extent of Shh signaling can indeed regulate the extent of foliation in mice.

In all of our mutants, the pattern of foliation reflects the normal progression of fissure formation during mouse development and represents a variation of patterns observed in different organisms. Of likely relevance to this, some of the tertiary fissures in the mouse cerebellum normally form only in certain genetic backgrounds (Inouye and Oda, 1980), and notably they correspond with fissures always present in the rat cerebellum. Furthermore, we found that all but one of these variable fissures are present in transgenic mice with increased Shh signaling (Shh-P1 mice) and a new fissure forms in a conserved position when signaling is further increased (Shh-P1;Ptch1^+/- mice). An attractive interpretation of these findings is that the location of fissures is preordained by an as yet to be discovered genetic pathway, and that the number of fissures that form in a particular mammal is determined by the extent of Shh signaling. The level and length of Shh signaling will be determined by many factors, including the number of PCs that are produced, the level of Shh transcription in different lobules and expression of factors that enhance or attenuate Shh activity.

	ACKNOWLEDGMENTS

 
We are grateful to Drs G. Fishell, R. Sillitoe and M. Zervas for critical reading of the manuscript, to Dr C. B. Bai for spinal cord analysis of Gli2/ embryos, and to Dr Roy Sillitoe for the rat cerebellum section. We thank Dr A. P. McMahon for providing the Smo conditional mutant mice and Kirsten Mimberg for technical assistance. S.B. was partially supported by a post-doctoral fellowship from the DFG. This work was supported by a grant from the NICHD. A.L.J. is a HHMI investigator.

	Footnotes

 
Supplementary material

Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/9/1811/DC1

^* Present address: Howard Hughes Medical Institute and Department of Genetics, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536, USA

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