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First published online 30 August 2006
doi: 10.1242/dev.02556
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1 Ben May Institute for Cancer Research, The University of Chicago, 924 E. 57th
Street, Chicago, IL 60637, USA.
2 Department of Human Genetics, The University of Chicago, 924 E. 57th Street,
Chicago, IL 60637, USA.
3 Department of Pathology, The University of Chicago, 924 E. 57th Street,
Chicago, IL 60637, USA.
* Author for correspondence (e-mail: wdu{at}huggins.bsd.uchicago.edu)
Accepted 31 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cyclin D1, Cerebellar development, Medulloblastoma, Ptch1, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The conserved hedgehog signaling pathway plays critical roles in regulating
the proliferation and differentiation of cerebellar GNPs
(Wechsler-Reya and Scott,
2001). In vitro studies have shown that sonic hedgehog (Shh),
which is secreted by the Purkinje neurons of the cerebellum, can sustain GNP
proliferation and maintain their undifferentiated state
(Wechsler-Reya and Scott,
1999). Shh acts by binding to its receptor patched (Ptch1) on
target cells. In the absence of Shh, Ptch1 inhibits the signaling activity of
smoothened (Smo), leading to the production of repressor forms of the Gli
family of transcription factors. Binding of Shh to Ptch1 blocks the inhibitory
activity of Ptch1 on Smo, resulting in the production of activator forms of
the Gli transcription factors and increased expression of downstream genes
(Stecca and Ruiz i Altaba,
2005).
Deregulation of the hedgehog pathway has been linked to the development of
a number of cancers, including medulloblastoma
(Goodrich et al., 1997;
Hahn et al., 1996). Mice that
are heterozygous for Ptch1 exhibit spontaneous development of medulloblastoma
(Goodrich et al., 1997). These
tumors exhibit increased Shh signaling
(Goodrich et al., 1997), and
treatment of mice with medulloblastomas with inhibitors of Shh signaling is
sufficient to cause tumor regression
(Berman et al., 2002;
Romer et al., 2004).
Interestingly, recent studies revealed the presence of preneoplastic lesions
in young adult Ptch1+/- cerebella. These preneoplastic
lesions exhibit an expression profile intermediate between those of normal
proliferating GNPs and medulloblastomas
(Oliver et al., 2005),
suggesting that medulloblastomas in Ptch1+/- mice probably
develop from these lesions. In addition, recent studies of human
medulloblastomas have showed that only tumor cells positive for the stem cell
marker CD133 are capable of forming tumors in mouse xenografts
(Singh et al., 2004), and
these tumor initiating cells exhibit other characteristics of neural stem
cells, such as self-renewal, proliferation and the capacity to differentiate
(Singh et al., 2003). These
observations support a tumor stem cell model for the development of
medulloblastomas.
Cyclins D1 and D2, but not D3, are upregulated in response to Shh signaling
in cerebellar cells (Kenney and Rowitch,
2000). Both cyclins D1 and D2 are expressed in proliferating GNPs,
and their joint removal results in an exceptionally hypoplastic cerebellum
(Ciemerych et al., 2002),
indicating that cyclins D1 and D2 play crucial roles in the rapid postnatal
expansion of the GNP population. Interestingly, although removal of either
cyclin D1 or D2 alone does not affect in vitro proliferation of postnatal day
4 GNPs in response to Shh signaling
(Kenney and Rowitch, 2000),
removal of cyclin D2 alone is sufficient to cause mild cerebellar hypoplasia
(Huard et al., 1999), owing to
both decreased proliferation and increased apoptosis of GNPs. This result is
somewhat surprising as the D-type cyclins have very similar biochemical
functions and, in general, specific effects of individual mutations are
typically seen only in tissues where the other D-type cyclins are not
expressed at high levels (Ciemerych et al.,
2002).
The cerebellar phenotype of Ccnd2-/- mice could be due to a requirement for two D-type cyclins concurrently in the proliferation of GNPs, or it could be due to differences in the expression patterns of these two cyclins in the developing cerebellum. In this report, we show that early postnatal GNPs expressed only cyclin D1 while later GNPs expressed both cyclins D1 and D2. Consistent with this expression data, Ccnd1-/- mice had decreased early GNP proliferation and early ataxia as a consequence of a delay in acquiring normal cerebellar function. Interestingly, removal of cyclin D1 resulted in a significant decrease in the incidence of medulloblastoma in Ptch1+/- mice, as well as a decrease in the incidence and size of preneoplastic lesions. We suggest that cyclin D1 plays a crucial role in the proliferation of early GNPs and in the progression of the preneoplastic lesions to medulloblastomas.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For
the righting reflex, five litters were tested daily beginning on P6. Mice were
turned on their backs and observed to determine whether they could immediately
right themselves. Ptch1+/-;Ccnd1+/-
mice were bred to obtain Ptch1+/-
(Goodrich et al., 1997) and
Ptch1+/-;Ccnd1-/- mice. All mice were
maintained on a mixed C57Bl/6 x 129Sv background. Mice used for the
tumor study represent six generations beginning with the F2 generation. Mice
were observed daily, and mice older than 35 weeks or demonstrating abnormal
neurological symptoms were euthanized and brains were dissected under a
dissecting microscope to examine for tumors. All mice were housed in a
facility accredited by the Association for Assessment and Accreditation of
Laboratory Animal Care and maintained in accordance with NIH Guidelines. The
Animal Care and Use Committee at The University of Chicago approved all
procedures.
Immunohistochemistry
Antibodies used: BrdU (BD Biosciences B44 1:800), cyclin D1 (Santa Cruz
sc-450 1:400), cyclin D2 (Lab Vision Ab-4 1:500), NeuN (Chemicon MAB377
1:40,000), calbindin (Swant 1:6000) and phosphohistone H3 (Upstate 1:16,000).
Samples were fixed, dehydrated and embedded in paraffin. Sections (6 µm)
were rehydrated, and endogenous peroxidases were blocked by incubation in 3%
H2O2, 10% methanol for 30 minutes. Antigen retrieval for
all antibodies was performed by boiling in 0.01 M citric acid (pH 6) for 10
minutes in the microwave. The primary antibody was detected using the Vector
Elite ABC kit (Vector Laboratories). Slides were lightly counterstained with
Hematoxylin and Eosin. Sections incubated without primary antibody served as a
negative control. For comparison of staining patterns within a single
cerebellum, serial sections were examined. At least three pups from different
litters were analyzed.
BrdU incorporation and statistic analysis
To monitor S-phase, mice were injected with BrdU (Sigma) (100 mg/kg body
weight) 1 hour prior to euthanasia. For statistical analysis of tumor
incidence, the Chi-squared test was used. For comparison of proliferation and
number of preneoplastic lesions, the Student's t-test was used.
All developmental sections examined for size, staining patterns and proliferation were midline sagittal sections of Ccnd1+/+ and Ccnd1-/- littermates. The midline was identified by the absence of cerebellar peduncles and fastigial nucleus. Multiple sections have been examined but only a single midline section was used to calculate the area. The area was determined by taking pictures with a Zeiss digital camera with a known pixel/µm ratio. The area in pixels was determined in Adobe Photoshop and converted to µm2. For quantification of proliferation, the number of BrdU and H3-P positive EGL cells per mm of EGL was counted. BrdU-positive cells were counted on midline sections in the same location in the anterior at P0 and at P6. As there were many fewer H3-P positive cells at P0, the H3-P positive cells over the entire length of the midline sections were counted to minimize variations. Statistical analysis used the paired two-tailed Student's t-test.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Similarly, very few cyclin D2 positive nuclei were detected
in the EGL at P0 (Fig. 2G,H).
At P6, cyclin D2 was detected throughout the outer zone of the EGL in an area
that was broader than that of cyclin D1
(Fig. 2I,J), although staining
was not uniform: a subpopulation of cells, generally towards the middle of the
EGL, expressed much stronger cyclin D2 levels. Furthermore, unlike cyclin D1,
nuclear cyclin D2 staining was still present in some of the EGL cells by P12
(Fig. 2K,L, arrows). These
immunohistochemistry results were further supported by western blot analysis
of total cerebellar extracts, which showed very low levels of cyclin D2 at P0
and much higher levels at P6, while high levels of cyclin D1 were present at
both time points (see Fig. S1 in the supplementary material).
To determine whether cyclin D2 was upregulated to compensate for the loss
of cyclin D1, we evaluated the levels of cyclin D2 at P0 in
Ccnd1-/- cerebella. Comparison of
Ccnd1-/- and wild-type siblings showed no difference in
the cyclin D2 staining pattern (see Fig. S2 in the supplementary material). As
Ccnd1-/-;Ccnd2-/- mice show severe
cerebellar hypoplasia with only slight upregulation of cyclin D3
(Ciemerych et al., 2002), it
was unlikely that cyclin D3 would be significantly upregulated in
Ccnd1-/- cerebella. Consistent with this, we were unable
to detect cyclin D3 in either wild-type or Ccnd1-/- GNPs
(see Fig. S2 in the supplementary material). Therefore, the absence of cyclin
D1 does not lead to an upregulation of cyclins D2 or D3 in the EGL.
Ccnd1-/- mouse cerebellar phenotype
Given the absence of high levels of D-type cyclins in the EGL during early
cerebellar development in Ccnd1-/- mice, we wondered
whether they might have a cerebellar phenotype. Ccnd1-/-
mice exhibit several symptoms such as early lethality, decreased size and a
hindleg clasping reflex that have long been hypothesized to reflect a
neurological defect (Sicinski et al.,
1995), but no specific histological deficiency has been reported.
To test if Ccnd1-/- mice might have a mild ataxia related
to their cerebellar development, we examined the righting reflex of
Ccnd1-/- pups (Aruga et
al., 1998; Crawley,
1999). Wild-type pups could right themselves by P8, but
Ccnd1-/- pups were unable to turn over until at least P14,
and in some pups the reflex was delayed until as late as P18. After this
point, the Ccnd1-/- adults displayed no obvious ataxia,
suggesting that the cerebellar functional defect had been corrected or
compensated for.
Examination of the Ccnd1-/- cerebellum indicated no gross abnormalities in foliation or organization of cellular layers at any age (data not shown). However, cerebellar cross sections did appear smaller than normal, so the sizes of Ccnd1-/- cerebella were compared with those of their littermates. At early time points the cerebellum was too small to be accurately dissected and weighed, so we estimated the size by measuring the area of a midline histological section. At P0 we found no significant difference in the cross-sectional area of the midline cerebellum (Fig. 3A,D); however, by P6 the cerebellum was reduced in size to 68% of that of wild-type siblings (Fig. 3B,E,G). This relative size difference persisted but did not increase from P6 to P12 (Fig. 3C,F,G). By contrast, the total brain weight of Ccnd1-/- brains (including the cerebellum) was 84% and 80% of the Ccnd1+/+ littermate weight at P6 and P12, respectively. Therefore, the cerebellum was more severely affected than other brain regions. In addition, the difference in the cerebellar sizes differed in its timing and severity from that of the total body weight of Ccnd1-/- mice, which continued to fall behind that of their littermates from 72% at P6 to 53% at P12 (Fig. 3H).
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Removal of cyclin D1 affects early granule neuron precursor proliferation
The size defect of Ccnd2-/- cerebella was reported to
be due to both decreased entry into S phase and increased apoptosis of GNPs
(Huard et al., 1999). To
determine if increased apoptosis could contribute to the decreased granule
neuron population in Ccnd1-/- cerebella, we performed
TUNEL assays on both P0 and P6 cerebella. Both wild-type and
Ccnd1-/- cerebella showed very low levels of apoptosis,
and no significant difference was seen between them (see Fig. S3 in the
supplementary material). Therefore, there is no evidence that increased
apoptosis significantly contributes to the smaller granule neuron population
in Ccnd1-/- mice, although we cannot absolutely exclude
this possibility.
We then examined whether Ccnd1-/- GNPs had decreased
proliferation. As the D-type cyclins are known to be involved in the
transition through G1 (Sherr and Roberts,
2004), we expected this phase of the cell cycle to be lengthened
and the percentage of cells in S phase to be decreased. To identify cells in
S-phase, pups were injected with BrdU 1 hour prior to sacrifice. Midline
sections were labeled with anti-BrdU or anti-phospho-histone H3 (H3-P)
antibodies to measure S-phase or M phase cells, respectively. BrdU
incorporation and H3-P staining were observed throughout the entire
anteroposterior axis of the midline EGL at E16.5 and P0, except in the
innermost one to two cell layers (Fig.
4A,B,E,F; data not shown). By P6 when the thickness of the EGL had
greatly increased, both markers were found only in the outer half of the EGL
(Fig. 4C,D,G,H; data not
shown), consistent with known proliferation and migration patterns.
The pattern of BrdU incorporation was roughly similar between
Ccnd1-/- and wild-type littermates except that BrdU
incorporation was consistently decreased in Ccnd1-/-
cerebella at P0 (Fig. 4A,B,I).
Quantification of S phase cells in the same anterior region of the EGL at P0
revealed that Ccnd1-/- cerebella exhibit a significant
decrease in BrdU incorporation (Fig.
4I; P +/+0=427 cells/mm, P
-/-0=363 cells/mm; P=0.028). Interestingly,
this difference was not observed at P6, when both cyclins D1 and D2 are
expressed at high levels (Fig.
4C,D,I). There was similarly a trend in decreased mitosis in the
Ccnd1-/- EGL at P0
(Fig. 4E-F,J), although this
was not statistically significant (P=0.07) probably due to the small
number of mitotic cells as a consequence of the very short length of mitosis.
This modest (about 15%) decrease in BrdU incorporation could account for the
decrease in the granule cell population over 6 days of rapid proliferation.
For example, if early GNPs proliferate at a rate of 15 hours/cycle
(Yoshioka et al., 1985), there
could be 6x24/15 (i.e. 9.6) rounds of GNP proliferation between P0 and
P6. If each round of cell proliferation is decreased by 15%, this could lead
to the number of cells at P6 being decreased to as little as
(1-0.15)9.6x100 (i.e. 21%) of the normal number. Therefore, a
small decrease in proliferation over the course of multiple cell cycles can
have a profound effect on the cerebellar size. Taken together, these results
indicate that at P0, when cyclin D1 is the only highly expressed D-type
cyclin, GNPs are impaired in their ability to progress into the cell cycle in
the absence of cyclin D1. The observed defect in the G1/S transition of
Ccnd1-/- GNPs corresponds precisely to the appearance of
the cerebellar size defect seen between P0 and P6.
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). To
determine whether Purkinje cell dendrite development was altered in
Ccnd1-/- cerebella, we examined sections from pups at
different developmental stages. At P6, Purkinje cell dendrites have not
extended very far, and there is little difference between wild-type and
Ccnd1-/- pups (data not shown). At P9, when dendrites have
begun to extend but have not developed the more complex branches
characteristic of adult cerebella, staining with calbindin showed stunted
dendrites in Ccnd1-/- pups
(Fig. 5A,B). We further
examined the dendrites by confocal microscopy and found that, at P9,
Ccnd1-/- Purkinje cells had simplified disorganized
dendritic trees (data not shown). Interestingly, adult
Ccnd1-/- Purkinje cells looked normal
(Fig. 5C,D); therefore, the
timing of the Purkinje cell abnormality corresponds to the period of ataxia
for Ccnd1-/- pups. As Purkinje cells did not express
cyclin D1 at any of the postnatal time points examined, it is unlikely the
observed Purkinje cell defect represents a cell-autonomous event; more likely
it represents a response to abnormalities in other cell types such as the
GNPs. Comparison of Ccnd1-/- pups with siblings at P6, P12 and P15 showed similar decreases in EGL thickness (see Fig. S4 in the supplementary material), suggesting that GNPs were not accumulating in the EGL due to migration abnormalities. Therefore, we did not expect to see abnormalities in the radial glia along which they migrate. Consistent with this, staining with GFAP showed a normal density and orientation for these cells (data not shown). We conclude that the granule neuron proliferation defect is more likely to be due to a cell-intrinsic effect of cyclin D1 than to abnormal signals from the other cells such as the Purkinje cells or glial cells.
Decreased medulloblastoma incidence in Ptch1+/-;Ccnd1-/- mice
Ptch1+/- mice, in which the Ptch1 allele has
been replaced with the lacZ gene, have been reported to show a 15-20%
incidence of spontaneous medulloblastoma
(Goodrich et al., 1997;
Wetmore et al., 2000).
Transcriptional profiling has shown that cyclin D1 is among the genes most
robustly activated by Shh signaling in GNPs
(Lee et al., 2003;
Oliver et al., 2003), the cell
from which the tumors are believed to arise, and in vitro cyclin D1 protein
levels are more rapidly upregulated relative to other D-type cyclins in GNPs
in response to Shh (Kenney and Rowitch,
2000). The observation that cyclin D1 plays a crucial role in
normal early GNP proliferation prompted us to determine whether loss of cyclin
D1 might decrease medulloblastoma incidence in Ptch1+/-
mice.
Ptch1+/-;Ccnd1+/- mice were crossed
to generate both Ptch1+/-;Ccnd1+/+ and
Ptch1+/-;Ccnd1-/- mice. As had been
previously reported (Sicinski et al.,
1995), Ccnd1-/- pups showed high rates of
early lethality with an especially steep decline in survival during the first
5 weeks of life, with 54% of
Ptch1+/-;Ccnd1-/- pups surviving to 5
weeks. After this period, the rate of mortality was very low, allowing us to
examine medulloblastoma development in surviving adults. The peak incidence of
symptomatic medulloblastoma in Ptch1+/- mice occurs at
16-24 weeks of age (Wetmore et al.,
2000), and a similar incidence of tumors (7/23) was found in
asymptomatic mice in this age range
(Goodrich et al., 1997). All
the Ptch1+/-;Ccnd1-/- mice included in
our analysis (Fig. 6H) survived
within or beyond this time point.
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To exclude the possibility that we did not find tumors in adult Ptch1+/-;Ccnd1-/- mice because pups with tumors were selected against during earlier development, we examined eight Ptch1+/-;Ccnd1-/- cerebella from mice 2.5-3.0 weeks old by staining the brain for ß-gal activity and comparing them with Ptch1+/-;Ccnd1+/+ cerebella of the same age. Although we expected only about half of the Ptch1+/-;Ccnd1-/- mice of this age to survive to adulthood, we could find no major abnormalities in cerebellar patterns of ß-galactosidase activity. In fact, there appeared to be fewer regions of abnormal surface staining than in Ptch1+/-;Ccnd1+/+ cerebella. In conclusion, our results show that although not absolutely essential, cyclin D1 plays an important role in Shh signaling induced medulloblastomas.
Medulloblastomas from Ptch1+/- mice express both cyclins D1 and D2
As the biochemical activities of the D-type cyclins appear similar, it has
been hypothesized that the requirement for a specific cyclin in a specific
tissue type is due largely to the regulation of its expression. In mouse
breast tumor models, the inhibition of tumorigenesis in
Ccnd1-/- tissue has been linked to the ability of an
oncogenic signaling pathway to upregulate cyclin D1 specifically
(Yu et al., 2001). To
determine if the requirement for cyclin D1 in medulloblastomas in
Ptch1+/- mice could be explained by it being the only
D-type cyclin expressed, we assessed the levels of both cyclins D1 and D2 by
immunohistochemistry. We found that both cyclins D1 and D2 were highly
expressed in the eight
Ptch1+/-;Ccnd1+/+ tumors examined
(Fig. 6E,G). All tumors had a
large fraction of cells expressing cyclins D1 (39±12.6%) and D2
(42±9.2%). BrdU staining showed that these tumors were also highly
proliferative, with an average of 25% of cells showing BrdU incorporation
(Fig. 6F). Interestingly, the
single Ptch1+/-;Ccnd1-/- tumor that
developed had a similar percentage of cyclin D2 positive cells (40%)
(Fig. 6J) and was highly
proliferative (33% BrdU positive) (Fig.
6I). Thus, the absence of cyclin D1 did not impact tumor cell
proliferation at this stage. As there was no further increase in the level of
cyclin D2 in the Ptch1+/-;Ccnd1-/-
medulloblastoma, these observations indicate that cyclin D1 is not required
for medulloblastoma cell proliferation and suggest that it is more likely to
play a key role at an earlier stage in medulloblastoma development.
Decreased `preneoplastic' lesions in Ptch1+/-;Ccnd1-/- mice
Adult Ptch1+/- mice have been reported to maintain
ectopic clusters of cells at the cerebellar surface, and these have been
suggested to represent preneoplastic lesions
(Goodrich et al., 1997;
Kim et al., 2003;
Oliver et al., 2005). To
determine if removal of cyclin D1 affected the incidence of these
preneoplastic lesions, we dissected 3-week-old
Ptch1+/-;Ccnd1+/+ and
Ptch1+/-;Ccnd1-/- cerebella, and
examined sagittal sections every 200 µm for ectopic lesions. As expected,
in the five wild-type cerebella examined the EGL was entirely absent, and no
ectopic clusters of cells were observed. By contrast, all
Ptch1+/-;Ccnd1+/+ and some
Ptch1+/-;Ccnd1-/- mice had ectopic
clusters of cells above the molecular layer. We divided these clusters into
two types based on cell proliferation: one that was proliferative
(Fig. 7A-E) and the other that
was not proliferative (Fig.
7F-J), as determined by staining for BrdU or H3-P. Similar to
mature granule neurons in the IGL, all cells in the non-proliferative clusters
expressed high levels of the post-mitotic neural marker NeuN
(Fig. 7F,G), and these clusters
did not express cyclin D1 or D2 (Fig.
7I,J). As we observed similar small ectopic clusters of
non-proliferative granule neurons in adult (35-week-old)
Ptch1+/-;Ccnd1+/+ mice that did not
develop medulloblastomas, these non-proliferative clusters are unlikely to
represent preneoplastic lesions. Interestingly, no significant difference in
the incidence of these non-proliferative lesions was observed between adult
Ptch1+/-;Ccnd1+/+ and
Ptch1+/-;Ccnd1-/- mice
(Fig. 7K).
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).
These lesions expressed both cyclins D1 and D2
(Fig. 7D,E), and were only
weakly positive for NeuN (Fig.
7A,B), similar to medulloblastomas
(Fig. 6D). Unlike the normal
EGL (Fig. 4C,D), there was no
organization into outer proliferative and inner post-mitotic layers. In some
proliferative lesions, BrdU-positive cells were found relatively evenly
throughout the entire collection of cells (not shown), while in others
(Fig. 7C) there were areas of
abnormal organization with BrdU-positive cells at both the inner and outer
edges of the cluster. In these cases, comparison of serial sections showed
that non-proliferative regions were more likely to express NeuN
(Fig. 7B). There was also an
increased number of strongly NeuN-positive cells in the molecular layer below
the proliferative lesions (compare Fig. 7B
and 7G), suggesting that some cells from the lesion were
differentiating and migrating out of these clusters. Additionally, TUNEL
staining revealed apoptotic cells within these lesions that were not detected
in the normal mature granule layer (see Fig. S3 in the supplementary
material). Each section was examined for proliferative preneoplastic lesions. If a lesion appeared in the same folia in two adjacent sections, it was deemed to represent a single lesion, whereas if a lesion appeared distinctly anterior or posterior to a lesion in a neighboring section it probably represented a different lesion. The size of individual lesions varied widely, with some small lesions appearing in only two consecutive sections (therefore extending less than 600 µm in the mediolateral direction), while other large lesions spanned up to 11 sequential sections (up to 2400 µm). The volume of a lesion was estimated based on its average cross-sectional area and its mediolateral span. Ptch1+/-;Ccnd1-/- pups were found to have significantly fewer of these preneoplastic lesions (Fig. 7K). The average number of independent lesions was approximately half that of Ptch1+/-;Ccnd1+/+ mice, while the total volume of lesions per mouse was sixfold smaller. The average size of an individual lesion was also one quarter of that in Ptch1+/-;Ccnd1+/+ mice. Therefore, we conclude that cyclin D1 contributes to the growth of preneoplastic lesions in young Ptch1+/- mice and that the decrease in the number of these cells probably accounts for the decreased incidence of medulloblastoma in Ptch1+/-;Ccnd1-/- mice.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and that Ccnd2-/- cerebella began to
show phenotypes after P3 (Huard et al.,
1999). These reports are consistent with our cyclin D2 expression
data and our suggestion that cyclin D2 can compensate for the absence of
cyclin D1 at P6.
Although both early and late GNPs can proliferate, it is possible that they
have distinct capacities to be transformed. For example, early GNPs
potentially behave more like stem cells and are therefore more susceptible to
transformation, while later GNPs have a more limited proliferation potential
because they are more committed to differentiation. In support of this idea,
it was reported that there was a gradual decrease in the ability of
irradiation to increase the incidence of medulloblastoma in
Ptch1+/- pups as they aged from P1 to P10: irradiation at
P1 or P4 increased medulloblastoma incidence to 81% or 51%, respectively,
while by P10 irradiation had no effect
(Pazzaglia et al., 2002;
Pazzaglia et al., 2006). This
is somewhat surprising given that there are many more GNPs at P4 than at P1,
so this difference probably reflects a greater susceptibility of early GNPs to
transformation.
An early role of cyclin D1 in medulloblastoma development
We propose a model that explains a role for cyclin D1 in medulloblastoma
development. Recently a preneoplastic stage of medulloblastoma was found to
exhibit gene expression patterns intermediate between those of GNPs and tumor
cells (Oliver et al., 2005),
suggesting that medulloblastomas are derived from these lesions. The
preneoplastic cells lacked expression of the wild-type Ptch1 allele,
suggesting that loss of Ptch1 expression is a rate-limiting step in
the transition from normal GNPs to preneoplastic lesions. We suggest that
genetic or epigenetic changes that activate Shh signaling in either early or
late GNPs could result in preneoplastic lesions with cells that have the
capacity to not only proliferate but also differentiate or die. However,
lesions derived from early GNPs also contain a population of cells that behave
more like stem cells and continue to renew, allowing them to acquire
additional genetic and epigenetic changes, which may lead to malignant
medulloblastoma. By contrast, transformation of late GNPs would be less likely
as these cells have a decreased proliferation potential and would require
re-activation of self-renewal pathways. This model predicts that removal of
cyclin D1, by limiting the proliferation of early GNPs, would reduce the
number of lesions arising from early GNPs. This is consistent with our
findings of an approximate halving in the number of independent preneoplastic
lesions. In addition, Ccnd1-/- early GNPs may be
intrinsically less competent for tumorigenesis, owing to their impaired
ability to proliferate. In support of this, we saw a more dramatic decrease in
the total volume of preneoplastic cells and in the incidence of
medulloblastomas in Ptch1+/-;Ccnd1-/-
mice.
Interestingly, while multiple preneoplastic lesions per mouse were observed
in young mice, only one or two lesions were observed by 6-8 weeks of age
(Oliver et al., 2005). These
results suggest that the majority of preneoplastic lesions will not progress
to tumors. As we detected both apoptosis and differentiation within these
preneoplastic lesions, it is likely that the abnormal cells in most of these
lesions either differentiate or undergo apoptosis, resulting in the
disappearance of the lesions. In addition to preneoplastic lesions, we found
that both young and adult Ptch1+/- mice had
non-proliferative clusters of cells, which were abnormally localized,
morphologically similar to mature granule neurons and strongly NeuN-positive.
We speculate that preneoplastic lesions might transition into these
non-proliferative clusters. In support of this, the preneoplastic lesions
often exhibited areas of non-proliferation with NeuN-positive cells.
This specific requirement for cyclin D1 during early tumorigenesis in the
Ptch1+/- medulloblastoma model contrasts with its role as
described in other tumor models (Robles et
al., 1998; Yu et al.,
2001), which revealed an important role for cyclin D1 only when
cyclin D1 was the only D-type cyclin upregulated in the tumor. Furthermore, in
the breast tumor model, continued cyclin D1/cdk4 kinase activity is required
for tumor cell proliferation (Yu et al.,
2006). By contrast, it does not seem likely that cyclin D1 is
required for proliferation of medulloblastoma cells as medulloblastomas that
developed in Ptch1+/-;Ccnd1+/+ mice
showed upregulation of both cyclins D1 and D2, and the one
Ptch1+/-;Ccnd1-/- medulloblastoma that
developed did not show a decreased level of proliferation.
Other cell cycle regulators in GNPs and medulloblastomas
During granule cell development, a number of cell cycle regulatory
proteins, including cyclin D2 and the cell cycle inhibitors p18 and p27, are
expressed in at least a partially overlapping manner, and deletion of any one
is often sufficient to create a mild cerebellar phenotype. The Cip/Kip family
member p27 was reported to be the only member of the Cip/Kip family of Cdk
inhibitors expressed in granule cell precursors, and it is detected in the
inner two-thirds of the EGL and in the IGL at P7
(Miyazawa et al., 2000). In
addition to p27, at least one member of the Ink family of Cdk inhibitors,
p18ink4c, which inhibits cyclin D/cdk4 kinase activity, is
expressed in the pool of proliferating granule cell precursors by P5, although
it is absent from early EGL cells at E15.5
(Zindy et al., 2003). Given
the intriguing possibility from our results that medulloblastoma development
may involve early GNPs that are present at P0, it will be interesting to
define more precisely the spatiotemporal expression of other cell cycle
regulators and the signaling pathways that regulate them in the GNPs at this
stage. In addition, as cyclin D2 is not expressed in early GNPs and removal of
cyclin D2 affects later GNP proliferation and apoptosis, it will be
interesting to determine if removal of cyclin D2 affects the incidence of
medulloblastomas and the number or size of preneoplastic lesions.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/19/3929/DC1
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ACKNOWLEDGMENTS |
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0.03 when
comparing between +/+ and -/- littermates. (J) Quantification of H3-P
positive nuclei in the EGL at P0 (n=8, P=0.07) and P6
(n=4, P=0.22) shows a trend of decreased mitotic cells in P0
but not in P6 GNPs in the mutant.
