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 9 September 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Shh, Foliation, Proliferation, Patterning
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ß-galactosidase staining
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). ß-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 at
http://saturn.med.nyu.edu/research/dg/joynerlab.
Histology, immunohistochemistry and RNA in-situ hybridization
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 5 µm. For consistency, sections analyzed from the
vermis were limited to the most medial 100 µm. Histology was performed on
paraffin sections using standard procedures. For antibody staining on X-gal
stained sections, frozen sections were incubated in substrate for 2-4 hours
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/joynerlab.
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 600
µm 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.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 ß-galactosidase (ß-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 ß-gal activity were detected in the anterior and most posterior lobes by 4 hours 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.
|
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 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 expression.
Gli1 and Gli2 are expressed in specific cell types of the cerebellum
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. Interestingly, Gli1-lacZ expression was 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.
|
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 similar thickness.
|
|
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 Ruizi-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.
|
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 cerebellum begins to expand by P8 in Shh-P1 mice
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 apparent.
|
|
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 reduced.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The responsiveness of granule cells to Shh is regulated
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 cerebellum resembles an exaggerated version of the normal cerebellum. Specifically, the 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., 2000).
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 cerebellum development.
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.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.[Medline]
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.[Medline]
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.[CrossRef][Medline]
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.[Medline]
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.[CrossRef][Medline]
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.[Medline]
Herrup, K. and Wilczynski, S. L. (1982). Cerebellar cell degeneration in the leaner mutant mouse. Neuroscience 7,2185 -2196.[CrossRef][Medline]
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.[CrossRef][Medline]
McAndrew, P. E., Frostholm, A., Evans, J. E., Zdilar, D., Goldowitz, D., 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.[CrossRef][Medline]
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
121,3935
-3945.
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.[Medline]
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.
16,2865
-2878.
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.[Abstract]
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.[CrossRef][Medline]
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 298,827
-830.
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.[CrossRef][Medline]
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.[Medline]
Wang, V. Y. and Zoghbi, H. Y. (2001). Genetic regulation of cerebellar development. Nat. Rev. Neurosci. 2,484 -491.[CrossRef][Medline]
Wechsler-Reya, R. J. (2001). Caught in the matrix: how vitronectin controls neuronal differentiation. Trends Neurosci. 24,680 -682.[CrossRef][Medline]
Wechsler-Reya, R. J. and Scott, M. P. (1999). Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 22,103 -114.[Medline]
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.[Medline]
Related articles in Development: