Cerebellar development is a carefully orchestrated process that
produces an exquisitely foliated structure with a simple layered
cytoarchitecture. In mammals, the cerebellum is divided into
three regions with distinct anteroposterior (AP) foliation
patterns: a central vermis and two bilaterally symmetric
hemispheres. The most abundant neurons in the cerebellum, as
well as the entire brain, are the granule cells. Whereas Purkinje
cells and cerebellar interneurons originate in the ventricular
neuroepithelium, cerebellar granular cell precursors (GCPs)
arise from a germinal zone in the rhombic lip situated in dorsal
posterior rhombomere 1 (Altman and Bayer, 1997). The GCPs
begin to leave the rhombic lip at approximately embryonic day
(E) 13 and migrate over the cerebellar anlage to form the
external granule layer (EGL). Although the EGL is formed by
E15, GCPs in the EGL remain mitotically active until 2weeks
postnatal. Granule cells start to exit the cell cycle after birth
and as part of their differentiation program migrate internally
past the Purkinje cells to form the inner granule layer (IGL)
(Wang and Zoghbi, 2001). Over the course of the first two
postnatal weeks, cerebellar folia form, suggesting the increase
in granule cells is largely responsible for foliation. The process
of foliation begins with the formation of four principal fissures,
which divide the cerebellum into five cardinal lobes (Altman
and Bayer, 1997). As GCP proliferation continues, these lobes
expand and are further subdivided to give rise to the species-
specific foliation pattern observed in the mature cerebellum.
The fissures that divide the central cardinal lobe into lobes VI-
VIII are among the last to form in the vermis.
It has been shown that an interaction between Purkinje cells
and GCPs is important for granule cell proliferation and
foliation. For example, when Purkinje cells are ablated or in
mouse mutants that lack Purkinje cells, such as Lurcher and
Staggerer, the GCP population is diminished and foliation is
arrested (Caddy and Biscoe, 1979; Herrup, 1983; Sidman et
al., 1962; Smeyne et al., 1995; Wetts and Herrup, 1982). One
key GCP mitogen expressed in Purkinje cells is sonic hedgehog
(Shh), since it can induce proliferation of GCPs in culture, and
injection of Shh antibodies into the cerebellum reduces granule
cell proliferation (Dahmane and Ruiz-i-Altaba, 1999; Wallace,
1999; Wechsler-Reya and Scott, 1999). Shh signaling is also
involved in many other developmental processes, in particular
in regulating cell fate decisions (Ingham and McMahon, 2001;
Jacob and Briscoe, 2003). In the spinal cord Shh induces
specific ventral cell types in a concentration-dependent
manner, and in the limb Shh determines digit identity. Since
The cerebellum consists of a highly organized set of folia
that are largely generated postnatally during expansion of
the granule cell precursor (GCP) pool. Since the secreted
factor sonic hedgehog (Shh) is expressed in Purkinje cells
and functions as a GCP mitogen in vitro, it is possible that
Shh influences foliation during cerebellum development by
regulating the position and/or size of lobes. We studied how
Shh and its transcriptional mediators, the Gli proteins,
regulate GCP proliferation in vivo, and tested whether they
influence foliation. We demonstrate that Shh expression
correlates spatially and temporally with foliation.
Expression of the Shh target gene Gli1 is also highest in
the anterior medial cerebellum, but is restricted to
proliferating GCPs and Bergmann glia. By contrast, Gli2
is expressed uniformly in all cells in the developing
cerebellum except Purkinje cells and Gli3 is broadly
expressed along the anteroposterior axis. Whereas Gli
mutants have a normal cerebellum, Gli2 mutants have
greatly reduced foliation at birth and a decrease in GCPs.
In a complementary study using transgenic mice, we show
that overexpressing Shh in the normal domain does not
grossly alter the basic foliation pattern, but does lead to
prolonged proliferation of GCPs and an increase in the
overall size of the cerebellum. Taken together, these studies
demonstrate that positive Shh signaling through Gli2 is
required to generate a sufficient number of GCPs for
proper lobe growth.
Key words: Shh, Foliation, Proliferation, Patterning
Spatial pattern of sonic hedgehog signaling through Gli genes
during cerebellum development
JoMichelle D. Corrales1,2, Gina L. Rocco1, Sandra Blaess1,2, Qiuxia Guo1,* and Alexandra L. Joyner1,2,3,†
1Howard Hughes Medical Institute and Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, 540 First
Avenue, New York, NY 10016, USA
2Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA
3Department of Physiology and Neuroscience, New York University School of Medicine, 540 First Avenue, New York, NY 10016,
*Present address: Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA
†Author for correspondence (e-mail: email@example.com)
Accepted 9 September 2004
Development 131, 5581-5590
Published by The Company of Biologists 2004
Shh mutants die at birth, we have utilized a gain-of-function
approach to address the role of Shh in vivo during postnatal
development in regulating GCP proliferation and a possible
role in foliation.
Shh signaling is mediated by the Gli family of transcription
factors. In the spinal cord Gli2 is the primary activator of Shh
signaling, whereas Gli3 functions mainly as a repressor but is
also a weak activator (Bai et al., 2002; Bai and Joyner, 2001;
Bai et al., 2004; Persson et al., 2002). By contrast, in the limb
only Gli3 is required for digit patterning and to regulate a
normal level of proliferation (Litingtung et al., 2002; te
Welscher et al., 2002; Wang et al., 2000). An important
question, therefore, is whether Shh functions in the cerebellum
primarily by inhibiting the Gli3 repressor as in the limb, and/or
by inducing the activator Gli2. Due to the embryonic lethality
of Gli2 and Gli3 mutants, the in vivo requirements for these
two genes during postnatal cerebellum development have not
been addressed. Gli1 (Gli – Mouse Genome Informatics),
however, is not required for mouse development, although it
plays a redundant activator function with Gli2, which is
revealed only in Gli2 heterozygotes (Bai et al., 2002; Park et
al., 2000). Furthermore, unlike that of Gli2 and Gli3, Gli1
transcription is regulated by Shh signaling. In particular, all
transcription of Gli1 is absolutely dependent on induction of
Gli2 and Gli3 activators by Hh signaling (Bai et al., 2004).
Since Gli1 is a transcriptional target of Shh signaling, lacZ
expression in Gli-lacZ knock-in mice (Gli1lz/+) is a readout of
positive Shh signaling.
We utilized Gli1-lacZ mice to characterize the precise
spatial and temporal pattern of positive Shh signaling in
the developing cerebellum. Strikingly, Shh expression and
signaling (Gli-lacZ expression) in the developing vermis is
spatially patterned from E18 to P10 with highest levels in
anterior lobes (III-VIa) and the most posterior lobe (X). Both
Gli1 and Gli2 are primarily excluded from Purkinje cells, and
Gli expression is strongest in Bergmann glia and in the GCPs
in the outer layer of the EGL. Gli3 is expressed in most cell
types along the AP axis. We show that in the absence of Gli2
normal expansion of GCPs in the EGL is impaired, and
foliation is reduced at birth. Gli1-lacZ expression is
undetectable in Gli2 mutants, demonstrating that Gli2 is the
major activator required to transduce Shh-positive signaling in
the developing cerebellum. In support of this, the thickness of
the EGL appears normal in Gli3 mutants. In transgenic mice
overexpressing Shh in a normal pattern in the cerebellum, the
basic pattern of cerebellum foliation is maintained, although
the entire cerebellum is enlarged and the lobes that normally
express higher levels of Shh have an irregular IGL. In addition,
the EGL persists longer than normal in transgenics. This study
utilizes in vivo experiments to establish a role for positive Shh
signaling in regulating expansion of the cerebellar lobes by
regulating GCP proliferation, and demonstrates that Gli2 is a
required mediator for this signaling.
Materials and methods
Timing of embryos was determined by designating noon of the day a
vaginal plug was detected as E0.5. The day of birth was designated as
P0. Mouse lines were maintained on an outbred Swiss Webster
background. lacZ knock-in mice Gli1lz/+and Gli2lz/+ were genotyped
by staining ear punches or tails in β-galactosidase (Bai et al., 2002;
Bai and Joyner, 2001). Shh-P1 mice were genotyped by PCR
usingprimers 5′-GGTCGGCGACAACTCAATCG and 5′-GT-
GAGGGTCTCTCAGCGTATG. Mice were genotyped for the Gli1zfd
allele using PCR primers g1.5489 5′-TTGCAGCCAGGAGTTC-
GATT, g1.6027R 5′-AGGACCCTACCTTGACTTGACACC and
NeopmR 5′-AGACTGCCTTGGGAAAAGCG (provided by C. Bai).
Mice were genotyped for the Gli2zfdallele as previously described
(Mo et al., 1997).
Brains from P10 or later stages were dissected after intracardiac
perfusion of mice with PBS followed by 4% paraformaldehyde. All
brains were immersion fixed in 4% paraformaldehyde at 4°C for 30
minutes. Fixed tissue was cryoprotected in 30% sucrose overnight at
4°C and embedded in OCT (Tissue-Tek). β-gal activity was detected
in 10-14 µm frozen sections by incubation in X-gal solution at
37°C for 4-6 hours unless otherwise indicated. Sections were
counterstained in Nuclear Fast Red. Detailed protocols are available
Histology, immunohistochemistry and RNA in-situ
Embryonic and early postnatal brains were dissected and immersion
fixed in 4% paraformaldehyde overnight at 4°C. Brains collected after
P5 were collected after intracardiac perfusion and fixed in 4%
paraformaldehyde overnight at 4°C. Tissue was embedded in paraffin
according to standard methods and sectioned at 5µm. For consistency,
sections analyzed from the vermis were limited to the most medial
100µm. Histology was performed on paraffin sections using standard
procedures. For antibody staining on X-gal stained sections, frozen
sections were incubated in substrate for 2-4hours and then post-fixed.
Antibody staining was performed according to standard protocol. The
following primary antibodies were used: BLBP (1:1000 kindly
provided by N. Heintz), Calbindin (1:4000, Sigma), Calbindin
(1:4000, Swant), and PCNA (1:500, Santa Cruz). Goat-anti-mouse
and goat-anti-rabbit biotinylated secondary antibodies (Vector
Laboratories) were used. Staining was visualized using an ABC kit
(Vector Laboratories) and DAB substrate. RNA in-situ hybridization
on sections was performed using standard methods. Detailed
protocols are available at http://saturn.med.nyu.edu/research/dg/
Quantitation of external and inner granule layers
To quantify EGL thickness, high magnification images were taken of
medial sections from wild-type (WT) and mutant cerebella of E18.5
embryos. Boxes 600µm in length were placed anterior to the primary
fissure, in the presumptive central lobe, and posterior to the secondary
fissure. The number of GCPs contained in each box was counted on
three sections from the most medial 100 µm of each embryo. Data
was obtained from three embryos of each genotype. The area
encompassed by the IGL in saggital sections was calculated using
MetaMorph software. Three representative sections from the most
medial 100 µm were used from mutant and control littermates from
three different litters.
Shh signaling is dynamic and spatially patterned
along the AP axis during cerebellum development
Previous studies have shown Shh and components of the Shh
pathway to be expressed in the developing mouse cerebellum
around birth (Dahmane and Ruiz-i-Altaba, 1999; Wallace,
1999; Wechsler-Reya and Scott, 1999). However, the temporal
onset and progression of Shh signaling and Gli expression in
mouse has not been fully documented. In order to determine
Development 131 (22)Research article
5583 Spatial Shh/Gli signaling in cerebellum
when and where cells respond to Shh signaling in the
developing cerebellum, we used Gli1-lacZ expression as a
readout of positive Shh signaling. We utilized mice expressing
lacZ from the Gli2 locus to examine Gli2 expression, and
performed RNA in situ hybridization to analyze Gli3.
Strikingly, Gli1-lacZ expression was found to be spatially
restricted along the AP axis during foliation of the cerebellum.
Gli1-lacZ was first detected at E18.5 in the EGL and some
deeper cells in a restricted pattern in the anterior region of the
medial cerebellum and in the region where the most posterior
fissure begins to form (Fig. 1B). Gli1-lacZ expression was also
restricted to the anterior region of the EGL in lateral sections
(Fig. 1B, inset). At P5, Gli1-lacZ expression was detected
throughout the AP axis in the EGL and deeper layers, although
expression in the central lobes (VIb to IX) was weaker (Fig.
1F). Interestingly, as fissures formed in the central lobe to give
rise to lobes VI-VIII, Gli1-lacZ was detected in the central
region. Since the level of lacZ expression is inversely
proportional to the length of time required to detect β-
galactosidase (β-gal) activity, we compared X-gal staining after
short (4-6 hours) and long (overnight) incubations (Fig. 1 and
data not shown). The highest levels of β-gal activity were
detected in the anterior and most posterior lobes by 4hours of
incubation (data not shown). Gli1-lacZ does not appear to be
differentially expressed within each lobe, as any subtle
differences probably reflect the variable thickness of the EGL
during early lobe formation. By P28, when foliation is
complete and the EGL has been depleted, expression of Gli1
was strongest in the Purkinje cell layer (PCL), which also
contains Bergmann glia, and this expression was homogeneous
along the AP axis (Fig. 1J). Weak expression was also detected
in the IGL in lobes III to VIa, in the posterior half of lobe IX,
and in lobe X, but only after staining for 24 hours.
The expression of Gli1 could be due to a similar restricted
expression domain of Shh or because the response to Shh is
spatially restricted. To address this we analyzed Shh mRNA
expression and found it to correlate with the pattern of Gli1
expression during cerebellum development. In midsagittal
sections through the cerebellar vermis, Shh mRNA was first
detected at E18.5 in a deep layer, and was highest in the
developing rostral three lobes (III-VIa) and lobe IX, similar to
Gli1-lacZ (Fig. 1A). At P5, Shh expression could be discerned
in the PCL and although it was detected throughout the AP
axis, it was lowest in the central lobes VIb-IX (Fig. 1E). By
the adult stage, Shh mRNA was detected at uniform levels in
all Purkinje cells along the AP axis of the cerebellum, similar
to Gli1-lacZ in the PCL (Fig. 1I).
To determine which Gli proteins could be activating Gli1
transcription, we examined the expression of Gli2 using mice
expressing lacZ from the Gli2 locus, and Gli3 expression
was determined using RNA in-situ hybridization. Gli2-lacZ
expression was quite distinct from the pattern of Shh and Gli1-
lacZ. Cerebellar expression of Gli2 was detected by E15.5,
earlier than the onset of Gli1 and Shh (data not shown). At
E15.5 and E18.5, Gli2-lacZ was expressed without spatial
restriction in the EGL and deeper layers of the cerebellum (data
not shown and Fig. 1C). During later stages and through to the
adult, strong Gli2-lacZ expression was detected broadly in the
cerebellum (Fig. 1G,K).
RNA in situ hybridization with a Gli3 antisense cDNA probe
was carried out to determine the developmental profile of Gli3
expression in the cerebellum. In a similar way to Gli2, Gli3
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. β-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: 125µm in A,B,C,D; 250µm in E,F,G,H; 500µm in I,J,K,L.
was not spatially patterned and was expressed in the EGL
and the deeper layers at E18.5 (Fig. 1D). By P5,
expression was maintained at similar levels along the AP
axis in the EGL and deeper layers (Fig. 1H). Like that of
Gli1, Gli3 expression in the EGL was stronger in the outer
EGL (Fig. 1H, inset). In the adult, Gli3 was expressed
broadly in most layers and appeared homogenous along the AP
axis (Fig. 1L). Thus, Gli2 and Gli3 are broadly expressed
throughout cerebellum development and do not correlate with
the temporally and spatially restricted Shh and Gli1-lacZ
Gli1 and Gli2 are expressed in specific cell types of
A primary site of Shh signaling during cerebellum
development is clearly the EGL, which is divided into an outer
proliferative layer and an inner differentiating layer.
strongest in the outer layer of the EGL (Fig.
2A-C). Strong expression of both Gli1 and Gli2
was also observed in deeper layers, which could
be due to expression in migrating granule cells,
Purkinje cells, and/or Bergmann glia that are in
close proximity to the Purkinje cells. In order
to determine which cell types express Gli1 and
Gli2, cryosections from Gli1lz/+ and Gli2lz/+P5
and P10 mice were stained with X-gal and
subjected to immunohistochemical labeling
with cell-type-specific antibodies (Fig. 2 and
data not shown). Antibodies against BLBP,
NeuN and Calbindin were used to mark
Bergmann glia, differentiated granule neurons
and Purkinje cells, respectively.
Gli1-lacZ expression was
Development 131 (22)Research article
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: 50µm.
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: 200µm in
B,C,E,F; 80µm in G,H,I.
5585 Spatial Shh/Gli signaling in cerebellum
Expression of Gli1-lacZ and Gli2-lacZ in Bergmann glia
was demonstrated by coexpression of lacZ with BLBP at P5
and P10 (Fig. 2A,D and data not shown). In the adult, when
Gli1 expression is homogeneous along the AP axis, Gli1-lacZ
was maintained at high levels only in Bergmann glia (data not
shown). Gli2 expression in the adult was at similar levels in
Bergmann glia and other cell types. Antibody labeling to detect
Calbindin at P10 demonstrated that Gli1 is not expressed in the
Purkinje cells, suggesting that positive Shh signaling is non-
autonomous in the cerebellum (Fig. 2B). Consistent with this,
most Calbindin-positive cells also did not express Gli2 (Fig.
2E), except for a few Purkinje cells, primarily in the posterior
lobes (Fig. 1G, between arrowheads, 1K, lobe X). Antibody
staining for NeuN, which marks differentiating granule cells,
confirmed that Gli1 expression is highest in the outer EGL. The
cells in the innermost layer of the EGL and the differentiated
GCs in the IGL did not express high levels of Gli1-lacZ (Fig.
2C). By contrast, Gli2 was expressed at similar levels in the
inner and outer EGL and also in the IGL (Fig. 2F). Thus, Gli1
is downregulated soon after granule cells begin to differentiate,
whereas Gli2 is not.
Gli2 is required to generate a multi-layered EGL and
to promote cerebellum foliation at E18.5
To determine whether the Gli proteins are required for granule
cell proliferation, we analyzed Gli2 and Gli3 mutants, since
Gli1 mutants have a normal cerebellum (Park et al., 2000) (see
Fig. 8A). The thickness of the EGL appeared normal in Gli3
mutants (Fig. 3I), however we cannot conclusively address
Gli3 function in GCP expansion due to early patterning defects
in rhombomere 1 (S.B. and A.L.J., unpublished). A recent
study suggested that the E18.5 Gli2 mutant cerebellum has
abnormal foliation that is more pronounced posteriorly, but a
detailed analysis was not performed (Palma and Ruiz i Altaba,
2004). To further explore positive Shh signaling in GCP
proliferation and cerebellar foliation, we analyzed Gli2 mutant
mice at E18.5, since the mutants die at birth. This is shortly
after the time when a response to Shh signaling is first detected
by analysis of Gli1-lacZ. Although the cerebellum is quite
immature at E18.5, whole-mount analysis of brains from E18.5
embryos clearly revealed that the cerebellum was smaller in
Gli2 mutants (n=7) compared with normal littermates (Fig.
3A,D). Sagittal sections further showed an almost complete
lack of foliation in medial regions (Fig. 3B,E). Furthermore, in
comparison with the normal EGL in the anterior and posterior
regions, which contained five to eight cell layers at E18.5 (Fig.
3B,G), the Gli2 mutant EGL contained only two to four cell
layers (Fig. 3E,H). In the developing central lobe and in lateral
sections, where foliation initiates after birth, the thickness of
the EGL and shape of E18.5 Gli2 mutant cerebella was similar
to that of WT (Fig. 3B,E and data not shown). In order to
further address whether the EGL in Gli2 mutants is primarily
affected in the regions that receive a high level of Shh, we
quantitated the thickness by counting the number of cells in the
EGL in three regions along the AP axis (see Materials and
methods and Fig. 3J). In areas where Gli1-lacZ is expressed,
indicated schematically by regions I and III in Fig. 3J, the
mutant EGL was reduced to 50-60% (P<0.0001) of the WT
EGL. However, in the central lobe, region II, where Gli1-lacZ
was not detected at E18.5, the WT and mutant EGL had a
We next used RNA in situ hybridization with EGL markers,
Math1 (Atoh1 – Mouse Genome Informatics) and Pax6, to
confirm that the cells remaining in the Gli2 mutant EGL were
in fact GCPs (Fig. 3F and data not shown). In addition,
antibody staining for PCNA, a marker for proliferating cells,
was performed to verify that the cells in the mutant EGL were
indeed proliferating (Fig. 3G,H). Importantly, at E16.5, the
EGL and size of the cerebellum in Gli2 mutants were
indistinguishable from those of normal embryos on sections
(data not shown), consistent with the appearance of a
phenotype in concert with the onset of Shh signaling in GCPs
In order to address whether cell types other than GCPs were
altered in Gli2 mutants, since one in vitro study indicated Shh
induces differentiation of Bergmann glia (Dahmane and Ruiz-
i-Altaba, 1999), immunohistochemistry was performed on
paraffin sections from E18.5 WT and Gli2 mutant cerebella.
Antibody marker analysis using Calbindin (Fig. 4A,E) and
BLBP (Fig. 4B,F) demonstrated that both Purkinje cells and
Bergmann glia were present in Gli2 mutants. At this stage,
Purkinje cells are not laminated in a single cell layer. The
Purkinje cells in Gli2 mutants appeared more clustered than in
WT cerebella; however, this is probably due to the decreased
surface area of the mutants resulting from the decreased GCP
pool and lack of foliation. Therefore, the cerebellum phenotype
in Gli2 mutants at E18.5 is probably due to a lack of Shh
signaling to the EGL.
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:
Finally, Shh-positive signaling was assayed in Gli2 mutants
by analyzing Gli1-lacZ activity and Gli1 mRNA expression in
E18.5 Gli2–/–embryos. Whereas Gli1-lacZ expression was
obvious in the anterior cerebellum of E18.5 WT embryos (Fig.
4C), no Gli1-lacZ could be detected in Gli1lz/+; Gli2–/–cerebella
(Fig. 4G). In addition, RNA in-situ hybridization analysis of
Gli1 expression in Gli2–/–cerebella showed drastically reduced
levels of Gli1 (Fig. 4H) compared with those of WT (Fig. 4D).
Since Gli1 is a direct transcriptional target of positive Shh
signaling, these results demonstrate that Gli2 is the main
activator of Shh-positive signaling in the cerebellum.
Overexpression of Shh in the cerebellum of Shh-P1
transgenics produces larger lobes and an irregular
inner granule layer
To determine whether elevated levels of Shh can increase
proliferation of GCPs and induce alterations in cerebellar
foliation, we employed a gain-of-function approach utilizing
transgenic mice (Shh-P1) carrying a 100 kb P1 clone that
contains the entire Shh coding region and some regulatory
sequences (Riccomagno et al., 2002). Gross inspection of
cerebella from adult transgenics showed that they were larger
than normal, especially in the AP axis, but the basic foliation
pattern appeared intact (Fig. 5A,E). Histological analysis
confirmed this and revealed a thicker IGL, as well as larger
lobes in the vermis and hemispheres compared with WT
cerebellum (Fig. 5B,C compared with 5F,G). The IGL was
thickest in lobes III, IV, V and IX, which correlates with the
normal expression pattern of Shh. In addition, the IGL
surrounding the primary fissure was irregular with distinct
bulges. Measurements of the area occupied by the IGL in Shh-
P1 mutants (see Materials and methods), showed a 30% overall
increase compared with those of WT.
The Shh-P1 transgene is known to be missing some negative
regulatory elements, since misexpression is observed in the
inner ear (Riccomagno et al., 2002). To determine whether the
transgene results in ectopic and/or overexpression of Shh in the
cerebellum, we utilized Gli1lz/+mice as a functional readout of
Shh signaling. X-gal staining of sagittal sections from P0 and
P5 Gli1lz/+; Shh-P1 double transgenic brains revealed an
increased intensity of X-gal staining in the mutant cerebellum
(Fig. 5D,H). Importantly, the labeling appeared in an anteriorly
restricted pattern identical to the pattern in WT mice (Fig.
5D,H and data not shown). Therefore, the transgene drives
overexpression in regions of endogenous Shh expression rather
than ectopic expression.
The cerebellum begins to expand by P8 in Shh-P1
In order to determine when and how the Shh-P1 phenotype
arises, transgenic brains were collected and sectioned from early
postnatal to adult stages. At P2 (data not shown) and P5 (Fig.
6A,E), the overall morphology of the Shh-P1 cerebellum (n=5)
was similar to that of normal (n=5) littermates (Fig. 6A). If Shh
normally elicits a proliferative response in GCPs in the EGL,
then an elevated level of Shh signaling might result in thickening
of this layer. However, analysis of sections at high magnification
did not reveal an obvious difference in the thickness of the EGL
in P5 mutant cerebella compared with WT. Consistent with this,
labeling with PCNA showed that at P5 in Shh-P1 transgenics
and WT littermates the thickness of the outer EGL was similar
(Fig. 7A,E). At P8, in contrast to P5, the IGL was thicker in Shh-
P1 brains than in WTs (n=4) (Fig. 6B,F). As with P5 Shh-P1
cerebella, however, the thickness of the EGL appeared normal.
By P14 (n=3) (Fig. 6C,G) and at P28 (n=4) (Fig. 6D,H), the
phenotype of transgenic mice was similar to that of adult mice
(n=5). The IGL was thickest and irregular in anterior regions
of the P28 cerebellum. These results show that the cerebellar
phenotype is first obvious when a compact IGL becomes
The IGL of P28 cerebella appeared more irregular than at P14,
suggesting the phenotype becomes more pronounced after P14.
One possibility was that depletion of the EGL is delayed, and
GCPs continue to proliferate longer in Shh-P1 transgenics. In
normal mice, most GCPs have differentiated and progressed into
the IGL by ~P14. By contrast, in Shh-P1 mutants the EGL
Development 131 (22) Research article
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: 400µm in B,C,F,G; 250µm in D,H.
5587 Spatial Shh/Gli signaling in cerebellum
contained three to four layers of cells at P14 (Fig. 7F), compared
with a single cell layer in normal mice (Fig. 7B). Furthermore,
the EGL in mutants persisted until P16 (Fig. 7H) when the EGL
in normal littermates was gone (Fig. 7D). We performed
antibody labeling with PCNA and found that both the WT and
mutant EGL contained proliferating cells at P14 (Fig. 7C,G) and
the EGL cells in the mutant continued to proliferate at P16 (data
not shown). Finally, Bergmann glia appear morphologically
normal and at normal density (data not shown).
The phenotype of Shh-P1 transgenics is sensitive to
the number of copies of endogenous Shh, but not Gli1
Since Gli1 transcription is upregulated in Shh-P1 transgenics,
we were interested in determining whether removal of this
activator of Hh targets could rescue the Shh-P1 phenotype. To
address this, Shh-P1; Gli1–/–mice were produced and compared
with Gli1 mutants, which have normal cerebella, and Shh-P1
single mutant mice. Based on whole-mount analysis (n=5, data
not shown) and histological sectioning (n=3; compare Fig. 8A-
C), Shh-P1; Gli1–/–mice appeared similar to Shh-P1 mice,
showing that removal of Gli1 does not rescue the mutant
phenotype. It is possible that the Gli2 activator is sufficient to
mediate positive Shh signaling in the absence of Gli1, but this
could not be addressed since Gli2 mutant mice die at birth.
We next tested whether the cerebellum phenotype was
sensitive to the number of copies of the endogenous Shh gene
by analyzing the cerebella of mice carrying the transgene and
heterozygous for a Shh null mutation (Shh-P1; Shh+/–n=3). Of
significance, removing one allele of Shh (Chiang et al., 1996)
partially rescued the Shh-P1 phenotype (compare Fig. 8B,D).
The partial rescue was variable between animals: two of four
animals displayed rescue at the level shown in Fig. 8D, whereas
the other two Shh-P1; Shh+/– mice only showed a slightly smaller
cerebellum than Shh-P1 mice. Histological analysis of Shh-P1;
Shh+/– cerebella showed that the IGL was not as thick or as
irregular as in Shh-P1 transgenics, and the overall size was
Shh expression and signaling is spatially patterned
It is of interest to determine how the pattern of fissures arises
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: 350µm in
A,E; 500µm in B,C,F,G; 400µm in D,H.
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: 50µm.
during cerebellum development in order to gain insight into the
relationship between development and function of particular
lobes. Our results demonstrate spatially patterned expression of
Shh and response to Shh signaling in the developing cerebellum
around birth. This raises the possibility that another mitogen is
responsible for inducing GCP proliferation early in the central
lobe of the cerebellum at birth. The positive response to Shh
signaling (Gli1-lacZ expression) in the developing anterior
vermis and lobe IX was observed in two cell types, the
proliferating GCPs in the outer EGL and the Bergmann glia in
the PCL. The division between high-level and low-level Gli1-
lacZ expression observed between lobes VIa and VIb correlates
with the expression borders of other genes that are anteriorly or
posteriorly restricted in the cerebellum, such as En-2, Fgf-1, and
Receptor protein tyrosine phosphatase ρ (McAndrew et al.,
1998; Millen et al., 1995). The same border is also identified in
Meandertail and Leaner mouse mutants, in which the anterior
lobes are specifically affected (Herrup and Wilczynski, 1982;
Napieralski and Eisenman, 1993; Ross et al., 1990). The spatial
pattern of Shh signaling during cerebellum development could
either simply reflect the intrinsic patterning of the cerebellum
along the AP axis, or reflect a direct role of Shh in the process.
Of possible relevance, at E18.5 expression of Gli1-lacZ and Shh
initiate at the same time the four principal fissures divide the
cerebellum into five cardinal lobes. In concert with the detection
in Gli1-lacZ and Shh in the central region of the cerebellum at
P5, fissures form to divide the central lobe into lobes VI-VIII.
Lobes V and IX undergo the greatest increase in length, and are
in areas that endure the longest temporal response to Shh
signaling. We propose that Shh signaling is not required for a
basal level of proliferation in the EGL, but is required to enhance
the level of proliferation required for lobe growth. In accordance
with this idea, [3H]thymidine labeling experiments to birthdate
GCs in the rat cerebellum demonstrated that the GCs in the
central lobes are the latest born, after those that comprise the
anterior and posterior regions (Altman and Bayer, 1997). We
found that, by overexpressing Shh in the normal domain, all the
lobes and IGL enlarge, with greatest effects in the anterior lobes.
Although it is not clear whether Shh regulates the position of
fissures, based on our studies we suggest that Shh regulates the
size and shape of the lobes by influencing the degree of GCP
The responsiveness of granule cells to Shh is
The observation that Gli1-lacZ is expressed at highest levels in
the outer EGL and Bergmann glia, and at low levels in the inner
EGL and IGL, demonstrates that the response of cells to Shh
signaling is precisely regulated. This response does not
correlate with their proximity to the source of Shh (Purkinje
cells), since Bergmann glia express high levels of Gli1-lacZ
and the immediately adjacent IGL cells express low levels.
Furthermore, Gli2 and Gli3 are broadly expressed in the
cerebellum, and therefore the response of cells to positive Shh
signaling is not regulated at the level of availability of the Gli
activators. Previous studies support a role for the extracellular
matrix (ECM) in regulating the proliferative response to Shh
(reviewed by Wechsler-Reya, 2001). GCPs in the outer EGL
are in contact with laminin, whereas differentiating granule
cells in the inner EGL and IGL are in contact with vitronectin.
Furthermore, GCPs proliferate extensively in the presence of
Shh when cultured on laminin, but not vitronectin. Although it
remains unknown how the ECM influences Shh signaling, the
spatial restriction of particular ECM molecules provides at
least one mechanism for the specific activation of Shh signaling
we observed in GCPs in the outer EGL.
A mechanism for Shh to elicit a proliferative response in
GCPs is by inducing the proto-oncogene Nmyc, which has been
shown to be a direct target of Shh (Kenney et al., 2003). Similar
to Gli1-lacZ, Nmyc is expressed in the proliferative outer EGL.
In accord with the mitogenic role of Shh in the cerebellum,
conditional mutant mice in which Nmyc is deleted in the
neuroepithelium display severe cerebellar hypoplasia due to a
reduced population of neuronal progenitors (Knoepfler et al.,
2002). However, these mutants lack Nmyc during early
formation of the cerebellar anlage; therefore, the GCP pool
may be compromised before Shh activity is required for later
expansion of the EGL.
Overexpression of Shh causes expansion of the
folia and inner granule layer
Increased levels of Shh in the cerebella of Shh-P1 transgenics
result in overall enlargement of the cerebellum. In addition,
there is a greater thickening of the IGL and distinct bulges
in the anterior vermis, where Shh levels are highest and
maintained over the longest time period during development.
The basic foliation pattern, however, is intact. During
development, the morphology of the mutant cerebellum
appears normal until P8, the stage at which the IGL is first
densely populated and becomes tightly compact.
Interestingly, the Shh-P1
exaggerated version of the normal cerebellum. Specifically, the
cerebellum resembles an
Development 131 (22) Research article
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: 320µm.
5589 Spatial Shh/Gli signaling in cerebellum
primary and invariant fissures are elongated and more distinct
in the mutant, whereas the variable fissures seen in lobes V and
VI of some WT mice are exaggerated and consistently seen in
all Shh-P1 transgenics. By the adult stage in Shh-P1 transgenics
the IGL has abnormal bulges surrounding the primary fissure,
probably reflecting a greater increase in granule cells in areas
of highest levels of Shh signaling. The primary fissure increases
the most in length, and also forms in a region that responds to
high levels of Shh signaling over the longest period of time. In
Shh-P1 mutants, this region is subjected to an increased level
of Shh due to the transgene for a longer period of time than the
central region. Although PCNA staining and the outer EGL
appear normal during early cerebellar development in Shh-P1
transgenics, the increased number of granule cells that make up
the thicker IGL probably results from generating an increased
number of GCPs. Therefore, the phenotype of Shh-P1
transgenics suggests that the level of Shh signaling influences
the differential growth of each lobe.
The division of the EGL into a proliferative and non-
proliferative layer raises the issue of how the GCPs move from
one layer to the other. Movement could be coupled to
differentiation, or alternatively, proliferation in the outer EGL
could force cells into the inner EGL if the layer could not
expand in length indefinitely. In Shh-P1 transgenics, the outer
EGL thickness does not increase, although the overall length
of the EGL is expanded due to increased lobe size. This
suggests that GCPs differentiate normally even when exposed
to excess Shh. The granule cells in Shh-P1 transgenics move
properly into the inner EGL and subsequently into the IGL,
although perhaps at a faster rate since the overall size of the
IGL is increased by P8. This indicates that the mechanism by
which cells exit the cell cycle is intact in these mutants,
preventing the accumulation of cells in the EGL. We did,
however, find that the EGL persists in Shh-P1 transgenics for
at least two more days than usual, similar to mouse mutants
lacking the cell cycle inhibitor p27/Kip1 (Miyazawa et al.,
Although Gli1 expression is increased in transgenics,
removal of Gli1 was not sufficient to rescue the phenotype.
This is consistent with a previous study showing that removal
of Gli1 in a mouse model of medulloblastoma in which Shh is
overexpressed does not lower tumor incidence (Weiner et al.,
2002). Furthermore, Gli2, and not Gli1, is required to mediate
positive Shh signaling in the embryo (Bai et al., 2002).
Gli2 is the major activator downstream of Shh
required in the cerebellum
Our analysis of E18.5 embryos lacking Gli2 demonstrates a
requirement for Gli2 in the positive response to Shh signaling
in GCPs. First, Gli2 mutants display a reduction in EGL
thickness in the regions in which Shh is expressed and
diminished foliation at E18.5, shortly after the onset of Gli1-
lacZ expression, which marks the positive response to Shh
signaling. Second, the Gli2 mutant phenotype seems to be
specific to the EGL, as other cell types such as Bergmann glia
and Purkinje cells appear normal. Finally, Gli1-lacZ expression
is not detected in Gli2–/–; Gli1lz/+embryos, demonstrating that
Gli2 is the major activator of Shh signaling in the cerebellum.
Gli3 could play a role only as a weak activator, since Gli1
expression is very weak in Gli2 mutants. In support of this, the
EGL in Gli3 mutants is not thinner than normal. The presence
of an EGL in Gli2 mutants, although reduced, suggests that
positive Shh signaling is not required for a basal level of
proliferation, but induces a heightened level of proliferation.
Due to the perinatal lethality of mutations in Gli2, a conditional
knockout is required to determine the role for Gli2 in postnatal
In summary, our studies highlight a mechanism for Shh
signaling in the cerebellum that primarily modifies Gli2 into
an activator to induce GCP proliferation. Using gene
expression analysis and a gain-of-function study, we also
demonstrate that the positive response to Shh signaling in the
cerebellum does not occur homogeneously along the AP axis.
Both the pattern of expression of Gli1-lacZ in the vermis and
the phenotype of Shh-P1 mutants correlate with a compartment
border observed by other gene expression patterns and
mutations affecting the cerebellum. Furthermore, there is a
direct correlation between the temporal onset of fissure
formation in different cerebellar regions and the timing of
elevated levels of Shh signaling in particular areas. Thus,
regulating the level and spatial pattern of Shh may have
provided a means during evolution to produce a more complex
foliation pattern in higher mammals.
We are grateful to Dana Lee and Kasia Losos for technical
assistance and to Drs Chunyang Bai, Gordon Fishell and Mark Zervas,
and Sema Sgaier for critical reading of the manuscript. This work
was supported by a grant from the NICHD. A.L.J. is an HHMI
Altman, J. and Bayer, S. A. (1997). Development of the Cerebellar System: In
relation to its Evolution, Structure, and Functions. New York, NY: CRC Press.
Bai, C. B. and Joyner, A. L. (2001). Gli1 can rescue the in vivo function of
Gli2. Development 128, 5161-5172.
Bai, C. B., Auerbach, W., Lee, J. S., Stephen, D. and Joyner, A. L. (2002).
Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation
of the Shh pathway. Development 129, 4753-4761.
Bai, C. B., Stephen, D. and Joyner, A. L. (2004). All mouse ventral spinal
cord patterning by hedgehog is Gli dependent and involves an activator
function of Gli3. Dev. Cell 6, 103-115.
Caddy, K. W. and Biscoe, T. J. (1979). Structural and quantitative studies on
the normal C3H and Lurcher mutant mouse. Philos. Trans. R. Soc. Lond.,
B, Biol. Sci. 287, 167-201.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal,
H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in
mice lacking Sonic hedgehog gene function. Nature 383, 407-413.
Dahmane, N. and Ruiz i Altaba, A. (1999). Sonic hedgehog regulates the
growth and patterning of the cerebellum. Development 126, 3089-3100.
Herrup, K. (1983). Role of staggerer gene in determining cell number in
cerebellar cortex. I. Granule cell death is an indirect consequence of
staggerer gene action. Brain Res. 313, 267-274.
Herrup, K. and Wilczynski, S. L. (1982). Cerebellar cell degeneration in the
leaner mutant mouse. Neuroscience 7, 2185-2196.
Ingham, P. W. and McMahon, A. P. (2001). Hedgehog signaling in animal
development: paradigms and principles. Genes Dev. 15, 3059-3087.
Jacob, J. and Briscoe, J. (2003). Gli proteins and the control of spinal-cord
patterning. EMBO Rep. 4, 761-765.
Kenney, A. M., Cole, M. D. and Rowitch, D. H. (2003). Nmyc upregulation
by sonic hedgehog signaling promotes proliferation in developing cerebellar
granule neuron precursors. Development 130, 15-28.
Knoepfler, P. S., Cheng, P. F. and Eisenman, R. N. (2002). N-myc is essential
during neurogenesis for the rapid expansion of progenitor cell populations
and the inhibition of neuronal differentiation. Genes Dev. 16, 2699-2712.
Litingtung, Y., Dahn, R. D., Li, Y., Fallon, J. F. and Chiang, C. (2002). Shh
and Gli3 are dispensable for limb skeleton formation but regulate digit
number and identity. Nature 418, 979-983.
McAndrew, P. E., Frostholm, A., Evans, J. E., Zdilar, D., Goldowitz, D.,
5590 Download full-text
Chiu, I. M., Burghes, A. H. and Rotter, A. (1998). Novel receptor protein
tyrosine phosphatase (RPTPrho) and acidic fibroblast growth factor (FGF-
1) transcripts delineate a rostrocaudal boundary in the granule cell layer of
the murine cerebellar cortex. J. Comp. Neurol. 391, 444-455.
Millen, K. J., Hui, C. C. and Joyner, A. L. (1995). A role for En-2 and other
murine homologues of Drosophila segment polarity genes in regulating
positional information in the developing cerebellum. Development 121,
Miyazawa, K., Himi, T., Garcia, V., Yamagishi, H., Sato, S. and Ishizaki,
Y. (2000). A role for p27/Kip1 in the control of cerebellar granule cell
precursor proliferation. J. Neurosci. 20, 5756-5763.
Mo, R., Freer, A. M., Zinyk, D. L., Crackower, M. A., Michaud, J., Heng,
H. H., Chik, K. W., Shi, X. M., Tsui, L. C., Cheng, S. H. et al. (1997).
Specific and redundant functions of Gli2 and Gli3 zinc finger genes in
skeletal patterning and development. Development 124, 113-123.
Napieralski, J. A. and Eisenman, L. M. (1993). Developmental analysis of
the external granular layer in the meander tail mutant mouse: do cerebellar
microneurons have independent progenitors? Dev. Dyn. 197, 244-254.
Palma, V. and Ruiz i Altaba, A. (2004). Hedgehog-GLI signaling regulates
the behavior of cells with stem cell properties in the developing neocortex.
Development 131, 337-345.
Park, H. L., Bai, C., Platt, K. A., Matise, M. P., Beeghly, A., Hui, C. C.,
Nakashima, M. and Joyner, A. L. (2000). Mouse Gli1 mutants are viable
but have defects in SHH signaling in combination with a Gli2 mutation.
Development 127, 1593-1605.
Persson, M., Stamataki, D., te Welscher, P., Andersson, E., Bose, J.,
Ruther, U., Ericson, J. and Briscoe, J. (2002). Dorsal-ventral patterning
of the spinal cord requires Gli3 transcriptional repressor activity. Genes Dev.
Riccomagno, M. M., Martinu, L., Mulheisen, M., Wu, D. K. and Epstein,
D. J. (2002). Specification of the mammalian cochlea is dependent on Sonic
hedgehog. Genes Dev. 16, 2365-2378.
Ross, M. E., Fletcher, C., Mason, C. A., Hatten, M. E. and Heintz, N.
(1990). Meander tail reveals a discrete developmental unit in the mouse
cerebellum. Proc. Natl. Acad. Sci. USA 87, 4189-4192.
Sidman, R. L., Lane, P. W. and Dickie, M. M. (1962). Staggerer, a new
mutation in the mouse affecting the cerebellum. Science 137, 610-612.
Smeyne, R. J., Chu, T., Lewin, A., Bian, F., Crisman, S., Kunsch, C., Lira,
S. A. and Oberdick, J. (1995). Local control of granule cell generation by
cerebellar Purkinje cells. Mol. Cell. Neurosci. 6, 230-251.
te Welscher, P., Zuniga, A., Kuijper, S., Drenth, T., Goedemans, H. J.,
Meijlink, F. and Zeller, R. (2002). Progression of vertebrate limb
development through SHH-mediated counteraction of GLI3. Science 298,
Wallace, V. A. (1999). Purkinje-cell-derived Sonic hedgehog regulates granule
neuron precursor cell proliferation in the developing mouse cerebellum.
Curr. Biol. 9, 445-448.
Wang, B., Fallon, J. F. and Beachy, P. A. (2000). Hedgehog-regulated
processing of Gli3 produces an anterior/posterior repressor gradient in the
developing vertebrate limb. Cell 100, 423-434.
Wang, V. Y. and Zoghbi, H. Y. (2001). Genetic regulation of cerebellar
development. Nat. Rev. Neurosci. 2, 484-491.
Wechsler-Reya, R. J. (2001). Caught in the matrix: how vitronectin controls
neuronal differentiation. Trends Neurosci. 24, 680-682.
Wechsler-Reya, R. J. and Scott, M. P. (1999). Control of neuronal
precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 22,
Weiner, H. L., Bakst, R., Hurlbert, M. S., Ruggiero, J., Ahn, E., Lee, W.
S., Stephen, D., Zagzag, D., Joyner, A. L. and Turnbull, D. H. (2002).
Induction of medulloblastomas in mice by sonic hedgehog, independent of
Gli1. Cancer Res. 62, 6385-6389.
Wetts, R. and Herrup, K. (1982). Interaction of granule, Purkinje and inferior
olivary neurons in lurcher chimaeric mice. I. Qualitative studies. J. Embryol.
Exp. Morphol. 68, 87-98.
Development 131 (22)Research article