Medulloblastoma is a highly malignant pediatric brain
tumor(Zakhary et al., 2001). Despite significant
improvements in treatment regimens, medulloblastoma
remains one of the leading causes of cancer-related death
in children under 9 years of age. A deeper understanding of
the molecular basis of medulloblastoma is crucial for
improving the early diagnosis and treatment of this
Approximately 25% of medulloblastoma cases have
mutations in components of the Sonic hedgehog-Patched
signaling pathway (Corcoran and Scott, 2001; Ellison et al.,
2003). Sonic hedgehog (Shh) is a potent mitogen for cerebellar
granule cell precursors (GCPs), the cells from which
medulloblastoma is believed to arise (Wechsler-Reya and
Scott, 2001; Wechsler-Reya and Scott, 1999). Patched
functions as an antagonist of Sonic hedgehog signaling in most
tissues (Ingham and McMahon, 2001). People with mutations
in the patched gene develop Gorlin’s syndrome, a disease
characterized by basal cell carcinomas, skeletal defects and an
increased incidence of medulloblastoma (Hahn et al., 1996;
Johnson et al., 1996). Sporadic medulloblastomas also harbor
mutations in patched and other elements of the Shh pathway
(Lam et al., 1999; Raffel et al., 1997; Taylor et al., 2002).
Finally, mice heterozygous for mutations in patched develop
cerebellar tumors that resemble human medulloblastoma
(Goodrich et al., 1997; Hahn et al., 2000).
Although patched mutant mice are an important model for
medulloblastoma, the molecular and cellular basis of
tumorigenesis in these mice remains unclear. Homozygous
patched knockout mice die during embryonic development
with defects in the nervous system, the heart and other tissues
(Goodrich et al., 1997). Heterozygotes survive to adulthood,
but after 3 months of age, 14-20% develop medulloblastoma
(Goodrich et al., 1997; Wetmore et al., 2000). The status of the
wild-type patched allele in these tumors is controversial: some
studies have reported expression of wild-type patched in tumor
tissue (Romer et al., 2004; Wetmore et al., 2000; Zurawel et
al., 2000), whereas others have suggested that the wild-type
allele is epigenetically silenced (Berman et al., 2002).
Determining whether patched is lost – and when during
tumorigenesis this loss occurs – is crucial for understanding
the mechanisms of medulloblastoma formation.
Studies of patched mutant mice suggest that cerebellar
abnormalities precede the appearance of tumors. While one-
sixth of these animals develop medulloblastoma at 3-6 months
of age, more than half have regions of ectopic cells in their
cerebella at 4-6 weeks of age (Corcoran and Scott, 2001;
Goodrich et al., 1997; Kim et al., 2003). These cells resemble
normal GCPs in terms of morphology and location on the
Medulloblastoma is the most common malignant brain
tumor in children. It is thought to result from the
transformation of granule cell precursors (GCPs) in the
developing cerebellum, but little is known about the early
stages of the disease. Here, we identify a pre-neoplastic
stage of medulloblastoma in patched heterozygous mice, a
model of the human disease. We show that pre-neoplastic
cells are present in the majority of patched mutants,
although only 16% of these mice develop tumors. Pre-
neoplastic cells, like tumor cells, exhibit activation of the
Sonic hedgehog pathway and constitutive proliferation.
Importantly, they also lack expression of the wild-type
patched allele, suggesting that loss of patched is an early
event in tumorigenesis. Although pre-neoplastic cells
resemble GCPs and tumor cells in many respects, they have
a distinct molecular signature. Genes that mark the pre-
neoplastic stage include regulators of migration, apoptosis
and differentiation, processes crucial for normal
development but previously unrecognized for their role
in medulloblastoma. The identification and molecular
characterization of pre-neoplastic cells provides insight
into the early steps in medulloblastoma formation, and may
yield important markers for early detection and therapy of
Key words: Medulloblastoma, Brain tumor, Pre-neoplastic, Patched,
Hedgehog, Migration, Differentiation, Mouse
Loss of patched and disruption of granule cell development in a
pre-neoplastic stage of medulloblastoma
Trudy G. Oliver1, Tracy Ann Read1, Jessica D. Kessler1, Anriada Mehmeti1, Jonathan F. Wells1,
Trang T. T. Huynh2, Simon M. Lin3and Robert J. Wechsler-Reya1,*
1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
2Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA
3Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC 27710, USA
*Author for correspondence (e-mail: email@example.com)
Accepted 16 February 2005
Development 132, 2425-2439
Published by The Company of Biologists 2005
Development and disease
surface of the cerebellum, and have therefore been described
as remnants of the external germinal layer (EGL) from which
GCPs originate. However, the presence of these cells in mice
that are destined to develop medulloblastoma raises the
possibility that they may represent a pre-neoplastic stage of
tumorigenesis. Determining whether these cells are merely
normal cells that persist into adulthood, or whether they are
partially transformed cells on their way to becoming tumors
has important implications for our understanding of granule
cell development and tumorigenesis.
To gain insight into the early stages of medulloblastoma
formation, we have isolated ectopic cerebellar cells from
patched mutant mice and studied their molecular and
functional characteristics. Our studies demonstrate that these
cells share many properties with tumor cells: they express
markers of the granule cell lineage, they exhibit activation of
Shh target genes and they proliferate extensively in vitro. In
addition, we show that these cells (and tumor cells) completely
lack expression of the wild-type patched allele, suggesting a
mechanism by which these cells maintain active hedgehog
signaling. Finally, microarray analysis reveals that these cells
have a unique pattern of gene expression that more closely
resembles tumor cells than GCPs. Thus, it is likely these cells
represent a distinct, pre-neoplastic stage of tumorigenesis.
Genes that are differentially expressed at the pre-neoplastic
stage include regulators of cell migration, survival and
differentiation. Our studies suggest that loss of patched
expression and dysregulation of these processes may be crucial
early events in the development of medulloblastoma.
Materials and methods
Patched heterozygous mice (Goodrich et al., 1997) were obtained
from Matthew Scott’s laboratory at Stanford (CA, USA) and
maintained by breeding with 129X1/SvJ mice from the Jackson
Laboratories (Bar Harbor, ME). Math1-green fluorescent protein
(Math1-GFP) transgenic mice (Lumpkin et al., 2003) were provided
by Jane Johnson at UT Southwestern Medical Center (Texas, USA).
Math1-GFP/patched+/–mice were generated by crossing patched
heterozygotes with Math1-GFP mice, and then backcrossing to
Math1-GFP mice three times before further analysis. All mice were
maintained in the Cancer Center Isolation Facility at Duke University
To detect expression of β-galactosidase in intact cerebellum, tissue
was isolated from adult wild-type or patched+/–mice (6- to 12-weeks
old) and fixed in 4% paraformaldehyde (PFA) at 4°C. After fixation,
tissues were permeabilized in buffer containing 0.01% deoxycholate
and 0.02% IGEPAL CA-630 (both from Sigma, St Louis, MO, USA)
for 10 minutes. Tissues were washed and stained overnight with X-
gal reaction mixture containing 10 mM potassium ferrocyanide, 10
mM potassium ferricyanide, 0.4 mg/ml X-galactoside 5-bromo-
4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal, Sigma) in
dimethyl sulfoxide, and 1 mM MgCl2in phosphate-buffered saline.
To compare expression of β-galactosidase and GFP in sections from
Math1-GFP/patched+/–mice, cerebella were fixed in 4% PFA,
cryoprotected in 25% sucrose, embedded in Tissue Tek-OCT (Sakura
Finetek, Torrance, CA, USA) and cryosectioned sagittally at a
thickness of 10 µm. One set of sections (for detection of GFP) was
post-fixed for 10 minutes in 2% PFA and immediately mounted in
Fluoromount G (Southern Biotechnology Associates, Birmingham,
AL, USA). Adjacent sections were stained with X-gal as described
above, counterstained with Nuclear Fast Red (Vector Laboratories,
Burlingame, CA, USA) and mounted in Fluoromount G. Fluorescent
(GFP) and bright-field (X-gal) images were acquired using a Nikon
TE200 inverted fluorescent microscope and Openlab software
(Improvision, Lexington, MA, USA).
For histochemical analysis of neonatal cerebellum, pre-neoplastic
lesions and tumors, cerebella were fixed overnight in 10% formalin,
transferred to 70% ethanol, paraffin wax-embedded and sectioned at
5 µm. Sections were stained with Hematoxylin and Eosin (Sigma).
Isolation of granule cell precursors, pre-neoplastic cells
and tumor cells
Granule cell precursors (GCPs) were isolated from 7-day-old (P7)
patched+/–mice; pre-neoplastic cells were obtained from 6-week-old
patched mutants; and tumor cells were obtained from 10- to 25-week-
old patched mutants displaying physical and behavioral signs of
medulloblastoma. Cells were isolated from each source using a
protocol described in (Wechsler-Reya and Scott, 1999). Briefly,
cerebella were digested in solution containing 10 U/ml papain
(Worthington, Lakewood, NJ, USA) and 250 U/ml DNase (Sigma),
and triturated to obtain a cell suspension. This suspension was
centrifuged through a step gradient of 35% and 65% Percoll
(Amersham Biosciences, Piscataway, NJ, USA), and cells were
harvested from the 35%-65% interface. Cells were resuspended
in serum-free culture medium consisting of Neurobasal containing
B27 supplement, sodium pyruvate, L-glutamine and penicillin/
streptomycin (all from Invitrogen, Carlsbad, CA, USA), and counted
on a hemacytometer. Cells used for RNA isolation were centrifuged
and flash frozen in liquid nitrogen. For proliferation assays or
immunostaining, cells were plated on poly-D-lysine (PDL)-coated
tissue culture vessels and incubated in serum-free culture medium.
Flow cytometry and immunofluorescence
To detect β-galactosidase activity in isolated GCPs, pre-neoplastic
cells and tumor cells, cells purified as described above were stained
with fluorescein di-β-galactopyranoside (FDG, Marker Gene
Technologies, Eugene, OR, USA) for 2 minutes at 37°C. Cells were
washed, incubated for 30 minutes on ice and analyzed on a
FACSVantage SE flow cytometer (BD Biosciences, San Jose, CA). As
a control for non-specific FDG staining, GFP–cells were isolated by
fluorescence-activated cell sorting
GFP/patched+/–mice and stained in the same manner. These cells are
not hedgehog responsive, and therefore express low levels of the
mutant patched allele and low levels of β-galactosidase.
To detect expression of surface markers, cells were stained for 1
hour with primary antibodies, washed, stained for 30 minutes with
secondary antibodies, and then analyzed by flow cytometry. To detect
expression of intracellular markers, cells were plated (1 million
cells/well) on PDL-coated coverslips in 24-well plates, and allowed
to adhere for 4-6 hours before fixation with 4% PFA. Cells were
stained overnight with primary antibodies, washed, stained with
secondary antibodies for 2 hours at room temperature, and then
mounted in Fluoromount G. Immunofluorescence was detected using
a Nikon TE200 inverted microscope and Openlab software.
Antibodies used for flow cytometry and immunofluorescence
included the following: nestin and GFAP (both from BD-Pharmingen,
San Diego, CA, USA); O4, A2B5, polysialated (PSA)-NCAM and
Zic-1 (all from Chemicon, Temecula, CA, USA); TUJ1 (Covance,
Berkeley, CA, USA); and 13A4 anti-prominin/CD133 (a generous gift
of Wieland Huttner and Denis Corbeil, Max Planck Institute, Dresden,
(FACS) from Math1-
Cerebellar cells isolated as described above were resuspended in
serum-free medium (Neurobasal + supplements) and transferred to
PDL-coated 96-well plates, at a density of 2?105cells/well. Cells
Development 132 (10) Research article
2427 Neoplastic stage of medulloblastomaDevelopment and disease
were pulsed immediately with tritiated thymidine (methyl-[3H]-Td,
Amersham, Arlington Heights, IL, USA) and cultured for 18 hours.
Following culture, cells were harvested onto filters using a Mach IIIM
Manual Harvester 96 (Tomtec, Hamden, CT, USA) and the amount of
incorporated radioactivity was quantitated by liquid scintillation
spectrophotometry using a Wallac MicroBeta microplate scintillation
counter (Perkin Elmer, Boston, MA, USA).
RNA isolation and real-time RT-PCR
To isolate total cytoplasmic RNA from GCPs, pre-neoplastic cells and
tumor cells, snap-frozen cell pellets were lysed in buffer containing
0.5% IGEPAL CA-630, digested with Proteinase K, extracted with
phenol:chloroform:isoamyl alcohol, and precipitated with ethanol.
RNA was purified using RNeasy columns (Qiagen, Valencia, CA,
USA) and treated with DNase 1 (DNA-free, Ambion, Austin, TX,
USA) to remove genomic DNA. RNA concentration was determined
using the RiboGreen fluorescent dye (Molecular Probes, Eugene, OR,
USA) with a TD-700 fluorometer (Turner BioSystems, Sunnyvale,
For real-time RT-PCR analysis, first-strand cDNA was synthesized
using equivalent amounts of total RNA (0.1-1 µg) in a 20 µl reverse-
transcriptase reaction mixture (Invitrogen). Real-time PCR reactions
were performed in triplicate using a 25 µl mixture containing iQ
SYBR Green Supermix (BioRad, Hercules, CA, USA), water, primers
and 1 µl of cDNA. Gene-specific primers were used for: Nmyc, cyclin
D1, Gli1, Pax6, Unc5h3 (Unc5c), Atf3, osteopontin (Spp1), Bag3,
Foxf2, Klf4 and Neurod1; sequences for these are available upon
request. Real-time quantitation was performed using the BIO-RAD
iCycler iQ system (BioRad). Serial tenfold dilutions of cDNA were
used as a reference for the standard curve calculation. Raw data were
normalized based on expression of actin.
For analysis of wild-type and mutant patched expression, cells were
isolated from Math1-GFP/patched+/–mice as described above, and
then FACS-sorted to obtain pure populations of GFP+cells. RNA was
isolated using the RNAqueous-Micro kit (Ambion), DNase-treated,
quantitated, converted to cDNA and subjected to real-time PCR
analysis as described above. Primers used to amplify patched were as
follows: exons 2-3, 5′-GGC AAG TTT TTG GTT GTG GGT C-3′
(forward) and 5′-CCT CTT CTC CTA TCT TCT GAC GGG-3′
(reverse); and exons 7-9, 5′-CAT TGG CAG GAG GAG TTG ATT
G-3′ (forward) and 5′-GCA CCT TTT GAG TGG AGT TTG G-3′
Microarray hybridization and analysis
RNA from GCPs, pre-neoplastic cells and tumor cells (isolated as
described above, but not FACS-sorted), and from normal adult
cerebellum (not dissociated), was converted to cDNA using the
Superscript Choice cDNA kit (Invitrogen) and a T7-dT(24) primer
(Genset/Proligo, Boulder, CO, USA). cRNA was generated using a
T7-transcription/labeling kit from Enzo Life Sciences and hybridized
to Affymetrix U74Av2 chips (Affymetrix, Santa Clara, CA, USA).
Chips were scanned, and hybridization data were acquired using
Affymetrix Suite 5.0 software. Affymetrix CEL files were normalized
and quantified using Bioconductor software with the gcRMA model
to quantify gene expression levels (Gentleman and Carey, 2002).
Unsupervised principal components analysis (PCA) was used to
identify the relationships among normal adult cerebellum, GCPs, pre-
neoplastic cells and tumor cells based on expression profiles.
To identify genes that were differentially expressed among GCPs,
pre-neoplastic cells and tumor cells, supervised analysis was carried
out. A gene-by-gene analysis of variance (ANOVA) model with three
groups (GCP, pre-neoplastic, tumor) was used to fit the log2-
transformed intensities. To correct for multiple comparisons, the
nominal P-value was adjusted using the false discovery rate (Benjamini
and Hochberg, 1995). Genes were considered to be differentially
expressed if they satisfied all of the following criteria: a difference in
expression greater than 1.9-fold between any two groups; a maximum
absolute intensity difference larger than 32 units; and an adjusted P-
value <0.01. There were 118 genes that met these criteria. The
identities of differentially expressed genes were verified by integrating
data from the Affymetrix and Unigene databases. Gene functions were
determined using information from Gene Ontology, Unigene,
LocusLink and PubMed databases. Clustering was performed with
Cluster and Treeview (Eisen et al., 1998). All statistical analysis was
performed using R-1.7 software (Dalgaard, 2002). Results were
visualized with Spotfire 6.0 (Somerville, MA, USA).
Immunohistological validation of microarray genes
Tissues were processed as described for the X-gal histological staining
in the methods above for P7, 6-week-old, and 10- to 25-week-old
patched mutant mice with tumors. PFA-fixed frozen sections were
rehydrated in Tris-buffered saline and permeabilized with 2% Triton
X-100 (Sigma) for 10 minutes. Sections were stained overnight at 4°C
with rabbit polyclonal antibodies specific for Zic3 (Chemicon),
Necdin (Upstate, Waltham, MA, USA) or Hsp105 (Biovision,
Mountain View, CA, USA), or with mouse monoclonal antibodies
specific for Pax6 (R&D Systems, Minneapolis, MN, USA). Antibody
staining was detected using the EnVision+ Peroxidase-DAB system
(Dako Cytomation, Carpinteria, CA, USA), as described in the
manufacturer’s protocol. Sections were counterstained with Harris
Hematoxylin (Sigma) and mounted using Vectamount (Vector
Detection of ectopic cerebellar cells in adult patched
Patched mutant mice develop cerebellar tumors that resemble
human medulloblastoma. These tumors are located on the
surface of the cerebellum and express high levels of β-
galactosidase (Fig. 1C). [When the patched mutant allele was
generated, a portion of the patched gene was replaced with the
β-galactosidase coding sequence (Goodrich et al., 1997); thus,
staining with the β-galactosidase substrate X-gal reflects
increased expression of the mutant patched allele.] Tumors
occur in 15-20% of mice between 10 and 25 weeks of age.
However, at 3-6 weeks of age, before any of the mice exhibit
physical manifestations of tumor development, more than half
have foci of ectopic β-galactosidase-expressing cells on the
surface of their cerebellum (arrowheads in Fig. 1B). At early
ages, several foci are commonly found in each animal; by 6-8
weeks, most animals have only one or two.
To examine the morphological characteristics of these cells,
we sectioned cerebella from patched mutant animals and
stained them with Hematoxylin and Eosin. At postnatal day 7
(P7), the cerebellum of patched mutant mice contains granule
cell precursors (GCPs) on its surface and is indistinguishable
from the cerebellum of wild-type mice (compare Fig. 2A with
2D). But by three weeks of age, wild-type and mutant cerebella
are clearly different. In wild-type animals, all GCPs have
differentiated and migrated inward, and the surface of the
cerebellum consists mostly of neuronal processes (Fig. 2B,C).
By contrast, 3-week-old patched mutant mice often have
multiple regions of cells that have failed to migrate and instead
remain on the surface of the cerebellum (Fig. 2E). These cells
are densely packed and have a high nucleus:cytoplasm ratio
(properties shared by both GCPs and tumor cells). In addition,
staining with antibodies to the proliferation marker Ki67
reveals that a large percentage of these cells are in cycle (data
not shown). Foci of ectopic cells are also seen in 6-week-old
patched mutant animals (Fig. 2F), but are rarely observed
beyond 10 weeks of age; at that age, patched mutant mice
either show no cerebellar abnormalities or have large tumors
that encompass the outer surface of their cerebellum (Fig. 2G).
Thus, the majority of patched mutant mice have proliferating
cells on the surface of their cerebellum well before they
The fact that the ectopic cells in patched mutants resemble
tumor cells in terms of morphology, location and β-
galactosidase expression – and occur in animals that are
destined to develop tumors – suggests that they might represent
an early, pre-neoplastic stage of medulloblastoma. Further
studies of these cells (described below) strongly support this
hypothesis; we therefore refer to them as ‘pre-neoplastic cells’
in the remainder of the text.
Pre-neoplastic cells express markers of immature
Previous studies have suggested that medulloblastoma arises
from granule cell precursors (Buhren et al., 2000; Kadin et al.,
1970; Miyata et al., 1998). To determine whether pre-
neoplastic cells express markers of the granule cell lineage, we
crossed patched+/–mice with Math1-GFP mice (Lumpkin et
al., 2003). In the developing cerebellum, Math1 is a specific
marker for proliferating granule cell precursors (Ben-Arie et
al., 1996; Helms and Johnson, 1998). As GCPs cover the
surface of the cerebellum during the first 2-3 weeks of life, the
cerebellum of Math1-GFP mice is intensely fluorescent during
this period (Fig. 3A). By 6 weeks of age, all GCPs have
migrated inward and differentiated into mature granule
neurons; thus, GFP is not expressed in the cerebellum of
adult Math1-GFP mice (Fig. 3B). However, in Math1-
GFP/patched+/–mice, regions of ectopic GFP+cells are
frequently seen in the cerebellum at 6 weeks of age (Fig. 3C,E).
Tumors arising in these mice are also strongly GFP+(Fig. 3D).
These data suggest that pre-neoplastic cells and tumor cells
express Math1 and are derived from the granule cell lineage.
To confirm that the GFP-expressing cells in adult Math1-
GFP/patched+/–mice are the same as the ectopic β-
galactosidase-expressing cells described above (Fig. 1), we
sectioned cerebella from these mice and stained them with X-
gal. As shown in Fig. 4A-C, GFP+regions of cerebellum from
6-week-old mice show strong X-gal staining. Likewise, tumors
from Math1-GFP/patched+/–mice show similar patterns of
GFP expression and X-gal staining (Fig. 4D-F). Thus, in
Math1-GFP/patched+/–mice, pre-neoplastic cells and tumor
cells can be clearly identified by their expression of β-
galactosidase and GFP. To gain insight into the early stages of
tumorigenesis in patched mutant mice, we sought to isolate
pre-neoplastic and tumor cells, and study their molecular and
Isolation of pre-neoplastic cells and tumor cells
from adult patched mutant mice
To isolate pre-neoplastic cells and tumor cells from patched
mutant mice, we used a method originally developed for
purifying neonatal GCPs (Oliver et al., 2003; Wechsler-Reya
and Scott, 1999). This method selects for small, dense cells
without processes, and destroys the majority of process-
bearing neurons and glia that make up the normal adult
cerebellum. Thus, when we used it to isolate cells from 6-
week-old wild-type mice, we obtained an average of only 0.5
million cells per animal (n=10, range 0.2-0.9 million), and
none of these cells had a morphology similar to that of GCPs.
By contrast, when we performed this procedure on 6-week-old
patched mutant mice, we obtained an average of 4.3 million
GCP-like cells (n=43, range: 0.5-50 million) (Fig. 5A). Among
patched mutant mice, 84% had more than 0.9 million cells, the
highest number obtained from a wild-type mouse at this age.
At 10-25 weeks of age, 16% of patched mutant mice had
readily identifiable tumors. From these animals, we could
isolate 50-600 million GCP-like cells (Fig. 5B). The remaining
animals had no discernable abnormalities in their cerebellum,
and we never obtained more than 2 million cells from them.
These studies demonstrate that pre-neoplastic cells and tumor
cells can be isolated from the cerebellum of patched mutant
To verify that the cells we isolated from patched mutant
mice represent the β-galactosidase-expressing cells identified
by whole-mount staining of the cerebellum (Fig. 1), we stained
Development 132 (10)Research article
Fig. 1. The majority of patched mutant mice have ectopic cells in
their cerebellum before they develop tumors. Cerebella from a 6-
week-old wild-type mouse (A), a 6-week-old patched+/–mouse (B)
and a 12-week-old patched+/–mouse with a tumor (C) were fixed
and stained with X-gal. Tumors (arrow in C) are found in 15-20% of
mutant mice between 10-25 weeks of age; these tumors express high
levels of the mutant patched allele, which carries the β-
galactosidase gene. At earlier ages, >50% of patched mutant mice
have ectopic β-galactosidase-expressing cells (arrowheads in B).
Background X-gal staining was not detected in the cerebellum of
adult wild-type mice (A).
2429 Neoplastic stage of medulloblastomaDevelopment and disease
these cells with a fluorescent β-galactosidase substrate (FDG)
and analyzed them by flow cytometry. As shown in Fig. 5C,
the pre-neoplastic cells isolated from 6-week-old patched
heterozygotes and tumor cells isolated from 16-week-old
animals were 89-96% FDG+. Moreover, when cells were
isolated from Math1-GFP/patched+/–mice, 87% of pre-
neoplastic cells and 95% of tumor cells were found to be GFP+.
Thus, freshly isolated pre-neoplastic cells and tumor cells, like
their counterparts in situ, express high levels of β-galactosidase
To further examine the phenotype of these cells, we isolated
Fig. 2. Ectopic ‘pre-neoplastic’ cells resemble tumor cells. Cerebella were isolated from wild-type mice (A-C) and patched+/–mice (D-G) at 7
days (A,D), 3 weeks (B,E) 6 weeks (C,F) or 12 weeks (G) of age. Tissues were paraffin wax-embedded, sectioned and stained with
Hematoxylin and Eosin. At 7 days of age, wild-type and patched mutant cerebella are indistinguishable, with densely-packed GCPs on the
surface (arrowheads in A and D). Note the presence of ectopic cells on the outside of the cerebellum in adult patched mutant mice (asterisks in
E and F) but not in wild-type mice (B,C). These cells are present in the majority of patched mutants, and resemble tumor cells (asterisk in G) in
terms of size, morphology and location.
them and stained them with antibodies specific for various cell
types (see Table S1 in supplementary material). Consistent
with the expression of Math1-GFP, a large percentage of cells
in each population expressed neuronal markers (tubulin
βIII/TuJ1, polysialated NCAM and Zic1) (Miyata et al., 1998;
Yokota et al., 1996). A small number of cells in each
population expressed the oligodendrocyte marker O4 (Sommer
and Schachner, 1981), suggesting that some cells of this
lineage co-purify with GCPs in our isolation procedure. By
contrast, very few cells expressed markers of astrocytes (glial
fibrillary acidic protein) (Bignami et al., 1972) or neural stem
cells (nestin, CD133/prominin) (Sawamoto et al., 2001;
Weigmann et al., 1997). These findings support the notion that
pre-neoplastic cells are derived from the granule cell lineage.
Pre-neoplastic cells express elevated levels of
hedgehog target genes and proliferate in vitro
In patched heterozygotes, β-galactosidase activity is a reporter
for expression of the mutant patched allele. Because patched
is a target of the hedgehog pathway (Goodrich et al., 1996),
the β-galactosidase activity observed in pre-neoplastic cells
(Fig. 1B, Fig. 4C) suggests that these cells have increased
activation of the hedgehog pathway. To determine whether
these cells express elevated levels of other hedgehog target
genes, we performed quantitative (real-time) RT-PCR analysis
using primers specific for Gli1, cyclin D1 and Nmyc, genes
Fig. 3. Pre-neoplastic cells express the granule cell lineage marker
Math1. Cerebella from 7-day-old (A) and 6-week-old (B) Math1-
GFP transgenic mice, and from 6-week-old (C,E) and 18-week-old
(D) Math1-GFP/patched+/–mice, were photographed using a Leica
MZFLIII microscope equipped with SPOT camera and software.
Fluorescent and bright-field images were overlaid using Photoshop.
(D) Entire brain, with cerebellum (including tumor) at bottom. Note
the GFP-expressing (green) pre-neoplastic and tumor regions (C-E)
in patched mutant mice.
previously identified as hedgehog targets in GCPs (Kenney et
al., 2003; Kenney and Rowitch, 2000; Oliver et al., 2003;
Wechsler-Reya and Scott, 1999). As a reference for the level
of mRNA associated with hedgehog pathway activation, we
compared the levels of these genes to the levels in resting
granule cell precursors (cultured for 24 hours in the absence of
Shh). As shown in Fig. 6, freshly isolated GCPs, pre-neoplastic
cells and tumor cells all exhibit significantly elevated levels of
Shh target genes when compared with resting cells (3- to 5-
fold for Nmyc, 4- to 9-fold for cyclin D1, 2000- to 6000-fold
for gli1). For each gene, the levels in GCPs, pre-neoplastic cells
and tumor cells were comparable to or higher than the levels
in cells that had been stimulated with Shh for 24 hours (data
not shown). These data suggest that the Shh signaling pathway
is activated in pre-neoplastic cells.
In granule cell precursors, Shh pathway activation is
associated with proliferation (Kenney and Rowitch, 2000;
Wechsler-Reya and Scott, 1999). To determine whether pre-
neoplastic cells are proliferating, we measured thymidine
incorporation in these cells following isolation. As shown in
Fig. 6D, pre-neoplastic cells incorporate thymidine at levels
comparable to freshly isolated GCPs and tumor cells. Thus, the
pre-neoplastic stage is characterized by persistent hedgehog
pathway activation and proliferation.
Pre-neoplastic cells lack expression of wild-type
Our studies indicated that GCPs, pre-neoplastic cells and tumor
cells all exhibit hedgehog pathway activation and proliferation
when they are isolated. In GCPs, expression of Shh targets
reflects exposure to Shh in vivo prior to isolation (Kenney and
Rowitch, 2000; Wechsler-Reya and Scott, 1999). In tumor
cells, Shh pathway activation has been suggested to result from
silencing of the wild-type patched allele (Berman et al., 2002),
although some groups have reported persistent expression of
wild-type patched in tumors (Romer et al., 2004; Wetmore et
al., 2000; Zurawel et al., 2000). The status of wild-type patched
in pre-neoplastic cells has not been investigated. Our ability
to isolate pre-neoplastic cells and tumor cells to near-
homogeneity allowed us to examine patched expression in
these populations in the absence of contaminating (non-tumor)
Development 132 (10) Research article
Fig. 4. Co-expression of Math1 and β-
galactosidase in pre-neoplastic cells and
tumor cells. Cerebella were isolated from
6-week-old (A-C) and 12-week-old (D-F)
tumor bearing Math1-GFP/patched+/–mice.
Intact cerebella (A,D) were photographed
using a Leica MZFLIII microscope and then
fixed, frozen and cryosectioned. One set of
sections was mounted and photographed
using a fluorescent microscope to detect
GFP (B,E); adjacent sections were stained
with X-gal, mounted and photographed
under bright field (C,F). Note the
correlation between GFP (green, indicative
of Math1 expression) and X-gal staining
(blue, indicative of mutant patched
expression) in pre-neoplastic (B,C) and
tumor-containing (E,F) regions.
Fig. 5. Pre-neoplastic cells can be isolated from the cerebellum of patched mutant mice. Cells were isolated from the cerebellum of wild-type
and patched+/–mice by enzymatic dissociation followed by Percoll gradient centrifugation, and viable cells were counted after Trypan Blue
staining. (A) The average yield for 6-week-old wild-type mice was 0.53±0.25 million cells. For patched heterozygotes of the same age, the
average yield was 4.3 million cells, with 84% of animals having more than 0.9 million cells (the maximum number seen in wild-type mice).
(B) Among older patched mutants (10-25 weeks), 16% had tumors containing 50-600 million cells; the remainder had fewer than 2 million
cells. (C) Non-granule cell precursors (GFP–cells from neonatal Math1-GFP/patched+/–mice), pre-neoplastic cells and tumor cells were
stained with the fluorescent β-galactosidase substrate FDG and analyzed by flow cytometry. Relative fluorescence of non-GCPs (blue), pre-
neoplastic cells (pink) and tumor cells (purple) is shown. The horizontal black line indicates the range of fluorescence considered to be positive
(i.e. above background); 89% of pre-neoplastic cells and 96% of tumor cells exhibited fluorescence within this range.
2431 Neoplastic stage of medulloblastomaDevelopment and disease
We analyzed patched expression in GCPs, pre-neoplastic
cells and tumor cells by real-time RT-PCR. To distinguish
between wild-type and mutant patched transcripts, we used
two sets of primers: one derived from exons 7-9, which are
present in both the wild-type and mutant alleles, and another
derived from exons 2-3, which can only amplify sequences
present in the wild-type allele (see Fig. 7A). As shown in
Fig. 7B, transcripts containing exons 7-9 were comparably
expressed in GCPs, pre-neoplastic cells and tumor cells. By
contrast, transcripts containing exons 2-3 (wild-type patched)
were found in GCPs but were absent from the majority of pre-
neoplastic cells and tumor cells (Fig. 7C). Similar results
were seen using a pair of primers within exon 2. Overall, loss
of wild-type patched was observed in 6 out of 7 pre-
neoplastic samples and 13 out of 13 tumor samples, but was
never seen in GCPs or in normal adult cerebellum from
patched+/–mice (see Fig. S1 in supplementary material).
These results suggest that the Shh pathway activation and
proliferation seen in pre-neoplastic and tumor cells results
from de-repression of the pathway due to loss of patched.
Moreover, they indicate that loss of patched occurs at an early
stage of tumorigenesis, well before cells have committed to
becoming full-blown tumors.
Pre-neoplastic cells have a unique molecular
Together, the above data demonstrate that GCPs, pre-neoplastic
cells and tumor cells are remarkably similar in terms of
morphology, cell lineage, hedgehog pathway activation, and
proliferation. However, all patched mutant mice have GCPs,
whereas only a subset have detectable pre-neoplastic cells
and only 16% develop tumors. Thus, despite their similarities,
GCPs, pre-neoplastic cells and tumor cells must differ at a
molecular level. To identify molecular differences between
Fig. 6. Pre-neoplastic cells exhibit hedgehog
pathway activation and proliferation.
(A-C) Expression of hedgehog target genes.
RNA was purified from freshly isolated GCPs,
pre-neoplastic cells and tumor cells, and from
GCPs cultured for 24 hours in the absence
(resting) or presence (stimulated) of Sonic
hedgehog (3 µg/ml Shh-N). Equivalent amounts
of RNA were reverse transcribed and subjected
to real-time PCR analysis using primers for
Nmyc (A), cyclin D1 (B) or gli1 (C). Expression
levels were normalized to actin and divided by
the levels in resting GCPs to calculate fold
induction. Data represent means of three samples
of each cell type ±s.e.m. (D) Proliferation of
GCPs, pre-neoplastic cells and tumor cells. Cells
were pulsed with tritiated thymidine immediately
after isolation, and then cultured for 18 hours in
serum-free media before being harvested and
assayed for thymidine incorporation. Data
represent means of triplicate samples ±s.e.m.,
and are representative of 16 experiments.
Fig. 7. Pre-neoplastic cells do not express wild-
type patched. (A) Primers used to distinguish wild-
type and mutant patched transcripts. The predicted
structures of wild-type and mutant patched
transcripts are shown. In the mutant allele, a
portion of exon 1 and all of exon 2 have been
replaced with the β-galactosidase coding sequence
(lacZ). Thus, sequences within this region (detected
by primers a and b) should only be present in wild-
type transcripts. Sequences within exons 7 and 9
(detected by primers c and d) should be present in
both wild-type and mutant transcripts.
(B,C) Expression of wild-type and mutant patched.
RNA from FACS-sorted GCPs, pre-neoplastic cells
and tumor cells was subjected to real-time PCR
analysis using primers c and d (exons 7-9, B) or
primers a and b (exons 2-3, C). Expression levels
were normalized to actin. Data represent means of
three samples±s.e.m. Loss of wild-type patched
expression was observed in six out of seven pre-
neoplastic samples and 13 out of 13 tumor samples;
see Fig. S1 in supplementary material for details.
GCPs, pre-neoplastic cells and tumor cells, we compared gene
expression in these populations using DNA microarrays. RNA
from GCPs, pre-neoplastic cells and tumor cells was labeled
and hybridized to Affymetrix U74Av2 GeneChips, and data
were analyzed using Bioconductor software (Gentleman and
Carey, 2002) (see Materials and methods for details).
To determine how closely related these samples were in
terms of gene expression, we performed unsupervised
principal components analysis (PCA). When the analysis
included normal adult cerebellum (Fig. 8A), GCPs, pre-
neoplastic cells and tumor cells appeared highly similar to one
another and quite distinct from adult cerebellum. This is not
surprising, as adult cerebellum consists of postmitotic neurons
and glia of various lineages whereas GCPs, pre-neoplastic
cells and tumor cells are all proliferating cells derived from
the granule cell lineage. By contrast, when GCPs, pre-
neoplastic cells and tumor cells were compared directly to one
another, each population exhibited a unique pattern of gene
expression (Fig. 8B). Hierarchical clustering of the samples
(Fig. 8C) supported this conclusion, and indicated that pre-
neoplastic cells and tumor cells resemble one another more
closely than either population resembles GCPs. Thus, GCPs,
pre-neoplastic cells and tumor cells are readily distinguishable
at a molecular level.
To characterize the differences in gene expression between
GCPs, pre-neoplastic cells and tumor cells, we performed
analysis of variance (ANOVA). This analysis identified 118
genes whose expression differed more than 1.9-fold (with a
statistical confidence of P<0.01) between populations (see Fig.
9). Expression of 75 genes differed between GCPs and pre-
neoplastic cells, and 80 genes distinguished GCPs from
tumors. Only 34 genes changed between pre-neoplastic and
tumor cells, consistent with the notion that these populations
are more closely related to one another than to GCPs.
A large proportion of the differentially expressed genes were
associated with three biological processes: cell migration, cell
stress/apoptosis and differentiation (Table 1). Among the
regulators of migration were extracellular matrix molecules
(Osteopontin, Pleiotrophin, Procollagen IV), cell surface
receptors (Unc5h3, Protein tyrosine phosphatase receptor type
z) and transcription factors (Pax6). Genes associated with
protection from cell stress included the transcription factor
Atf3, the chaperone regulator Bag3, and several heat shock
proteins. Finally, genes involved in cell fate and differentiation
included transcription factors such as Foxf2, Klf4, Sox2,
Neurod1 and Zic3. Significant differences in expression were
also seen in genes encoding signaling molecules (G-protein
coupled receptor 37-like 1, Gab1, Rhon, Calmodulin-like 4),
metabolic genes (Carbonic anhydrase 2, Prostaglandin D2
synthase), channels (Aquaporin 4, Kcnd2), and secreted factors
(Chemokine ligand 27, Vascular endothelial growth factor C).
Finally, a number of sequences resembling endogenous
retrovirus-like elements known as intracisternal A particles
(IAPs) were found to be overexpressed at the tumor stage.
To confirm the results of our microarray analysis, we
isolated RNA from independent samples of GCPs, pre-
neoplastic cells and tumor cells and examined expression of
representative genes using real-time RT-PCR. As shown in Fig.
S2 (see supplementary material), each of these genes (Pax6,
Unc5h3, Atf3, Osteopontin, Bag3, Foxf2, Klf4 and Neurod1)
showed a distinct expression pattern in GCPs, pre-neoplastic
cells and tumor cells, and the changes in expression were
consistent with our microarray data. To test whether
differentially expressed genes were targets of the Shh pathway,
we cultured GCPs in the presence or absence of Shh protein
for 12-24 hours and analyzed expression of select genes by
real-time RT-PCR (data not shown). None of genes we tested
were significantly altered by Shh stimulation, supporting the
notion that the differences between GCPs, pre-neoplastic cells
and tumor cells are not the result of differential Shh pathway
activation. Finally, to determine whether the genes we
identified in purified cells were also differentially expressed in
situ, we used antibodies against the protein products of some
of these genes to stain sections of neonatal (P7) cerebellum,
pre-neoplastic lesions and tumors. As shown in Fig. 10,
expression of these proteins (Zic3, Pax6, Necdin and Hsp105)
was significantly different in GCPs, pre-neoplastic cells and
tumor cells in situ, and mirrored the expression of the
corresponding transcripts in purified cells.
Together these studies demonstrate that GCPs, pre-
neoplastic cells and tumor cells can be distinguished at a
molecular level and are likely to represent distinct stages of
Development 132 (10) Research article
Fig. 8. GCPs, pre-neoplastic cells and tumor cells have distinct gene expression profiles. Gene expression in GCPs (four separate litters), pre-
neoplastic cells (five mice), and tumor cells (five mice) from patched mutant mice, and adult cerebellum from wild-type mice (four mice), was
analyzed using Affymetrix U74Av2 microarrays. Unsupervised principal components analysis (PCA) was used to assess the similarity in gene
expression between these samples. (A) PCA plot of all 18 samples indicates that GCPs, pre-neoplastic cells and tumor cells are very similar to
one another when compared with normal adult cerebellum. (B) Analysis excluding normal adult cerebellum suggests that compared with one
another, GCPs, pre-neoplastic cells and tumor cells each have unique profiles of gene expression. (C) Single-linkage hierarchical clustering of
the samples suggests that GCPs, pre-neoplastic cells and tumor cells are distinct, with pre-neoplastic and tumor cells resembling one another
more closely than either resembles GCPs.
2433 Neoplastic stage of medulloblastomaDevelopment and disease
AF053550: metaxin 2
AW124268: RIKEN cDNA 2810484M10 gene
U82758: claudin 5 (lung specific membrane protein)
AW125526: RIKEN cDNA 0610038D11 gene
AB016602: G protein-coupled receptor 37-like 1
J05154: lecithin cholesterol acyltransferase
AI843164: DnaJ (Hsp40) homolog, subfamily B, member 10
M63801: gap junction membrane channel protein alpha 1 (connexin 43)
U34277: PAF acetylhydrolase
Y08135: sphingomyelin phosphodiesterase, acid-like 3A
X17320: Purkinje cell protein 4
AI843119: glutathione S-transferase omega 1
AJ133130: protein tyrosine phosphatase, receptor type, Z1 (ptprz, DSD-1 proteoglycan)
AW123564: RIKEN cDNA similar to homeobox only domain (Hod)
X61452: septin 4
U07235: aldehyde dehydrogenase 2, mitochondrial
AI847646: calmodulin-like 4
M11533: myelin basic protein
AI850090: RIKEN cDNA 5730469M10 gene
AB006361: prostaglandin D2 synthetase (brain)
M25944: carbonic anhydrase 2
Y07812: myelin and lymphocyte protein, T-cell differentiation protein
U52073: N-myc downstream regulated-like
AW230977: RIKEN cDNA 1110017N23 similar to glucuronyl C5-epimerase
M15832: procollagen, type IV, alpha 1
AF081789: complement component 1, q subcomponent, receptor 1
M74773: brain beta spectrin
U48398: aquaporin 4
U88623: aquaporin 4
Z49976: glutamic acid decarboxylase 1
AW046694: RIKEN cDNA 4631408O11 gene
X68837: secretogranin II
AF016482: ras homolog N (RhoN), Rho7
AF044672: synuclein, alpha
X63963: paired box gene 6
AI848668: sterol-C4-methyl oxidase-like
AJ002366: general transcription factor II H, polypeptide 1
AW124932: 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1
AA672499: chemokine (C-C motif) ligand 27
AB012693: CD47 antigen
AI848868: RIKEN cDNA similar to CD47
U73620: vascular endothelial growth factor C
AW120579: eomesodermin homolog
AI839615: potassium voltage-gated channel, Shal-related family, member 2
D70849: zinc finger protein of the cerebellum 3
AI842432: phosphoglucomutase 2
AF020313: amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein
U72634: unc-5 homolog C
AB017202: nidogen 2 (entactin-2)
AW124340: solute carrier family 39 (metal ion transporter), member 8
U04827: fatty acid binding protein 7, brain (brain fatty acid binding protein)
X13752: aminolevulinate, delta-, dehydratase
U35249: menage a trois 1 (CDK activating kinase assembly factor p36/MAT1)
AI839901: mitochondrial carrier homolog 2 (C. elegans)
AI846851: farnesyl diphosphate synthetase
L31777: triosephosphate isomerase
U28068: neurogenic differentiation 1
AF020039: isocitrate dehydrogenase 1 (NADP+), soluble
AL021127: NAD(P) dependent steroid dehydrogenase-like
AF004294: myelin transcription factor 1
AB031291: transgelin 3
AI854020: cysteine dioxygenase 1, cytosolic
AI046826: growth factor receptor bound protein 2-associated protein 1
AW106745: NAD(P) dependent steroid dehydrogenase-like
AI843448: microsomal glutathione S-transferase 3
AB016424: RNA binding motif protein 3
AF004666: solute carrier family 8 (sodium/calcium exchanger), member 1
X13986: secreted phosphoprotein 1 (osteopontin)
U19118: activating transcription factor 3
U19118: activating transcription factor 3
AI646638: frequently rearranged in advanced T-cell lymphomas 2
AI839138: thioredoxin interacting protein
U02098: purine rich element binding protein A
AW049360: RIKEN cDNA 1700017B05 gene
AI837625: cysteine and glycine-rich protein 1
D88793: cysteine and glycine-rich protein 1
AJ223087: cell division cycle 6 homolog
AW120868: EST similar to TCDD-inducible poly(ADP-ribose) polymerase (PARP7)
AV373612: Bcl2-associated athanogene 3
L40406: heat shock protein 105
AF060565: RW1 protein
AI047107: RIKEN cDNA 3732413I11 gene
AW124633: NIMA (never in mitosis gene a)-related expressed kinase 7
X53584: heat shock protein 1 (chaperonin, HSP60)
AI987985: zinc finger protein 288
M24377: early growth response 2 (Krox-20)
AB028272: DnaJ (Hsp40) homolog, subfamily B, member 1
AA592351: cDNA sequence BC025546
AF061260: immunoglobulin superfamily, member 4a (nectin-like protein 2)
Y12293: forkhead box F2
X75316: RNA-binding region (RNP1, RRM) containing 1 (seb-4)
X16670: type IIB intracisternal A-particle (IAP) element encoding integrase
X04120: intracisternal A particles, IL3 linked
AA980810: RIKEN cDNA 5730405M13 gene
AW049619: cyclin-dependent kinase 8
X51528: ribosomal protein L13a
X14897: FBJ osteosarcoma oncogene B
AW045753: RIKEN cDNA 1110015E22 gene
M21050: lysozyme M
U02880: keratin complex 1, acidic, gene 12
X07625: protamine 1
AI853172: UDP-glucose ceramide glucosyltransferase
X16672: type IIB intracisternal A-particle element encoding integrase and gag
V00830: keratin complex 1, acidic, gene 10
M17551: intracisternal A-particle gag protein
M10062: intracisternal A particle
AW209004: cDNA clone similar to Mouse IgE-binding factor mRNA
U20344: Kruppel-like factor 4 (gut) (GKLF)
AA111360: Echinoderm microtubule associated protein like 5 (nuclear protein UKp68)
AI746846: sorting nexin 10
SRY-box containing gene 2
melanoma inhibitory activity
log2 fold: 4
Fig. 9. Genes that distinguish
GCPs, pre-neoplastic cells and
tumor cells. Microarray data were
subjected to analysis of variance
(ANOVA) and genes that changed
more than 1.9-fold between
GCPs, pre-neoplastic cells and
tumor cells (with adjusted P-
values <0.01 and maximum
absolute intensity difference >32
units) were considered
Expression profiles of the 118
differentially expressed genes are
shown. Colors represent relative
expression level, with red
denoting high and green denoting
low expression (see gradient at
bottom of figure). Genes were
clustered based on expression
pattern among the three groups.
tumorigenesis. It is important to note that few of the genes that
were differentially expressed between GCPs, pre-neoplastic
cells and tumor cells were granule cell lineage markers,
elements of the cell cycle machinery or known targets of the
hedgehog pathway. This is consistent with our previous
observation that these cells resemble one another in terms of
lineage, hedgehog pathway activation and proliferation. By
comparing similar populations of cells at different stages of
tumorigenesis, we were able to identify other processes and
pathways that contribute to medulloblastoma formation. The
fact that pre-neoplastic cells and tumor cells have significant
changes in genes that control migration, apoptosis and
differentiation suggests that dysregulation of these processes
may be crucial for the development of medulloblastoma.
Medulloblastoma is a highly malignant and frequently fatal
tumor, but little is known about the early stages of the disease.
We have used a mouse model of medulloblastoma to identify
a pre-neoplastic stage and define its molecular characteristics.
Our data demonstrate that pre-neoplastic cells show elevated
hedgehog pathway activation and proliferation, and lack
expression of wild-type patched. In addition, pre-neoplastic
cells have a unique molecular signature characterized by
altered expression of genes that regulate migration, apoptosis
and differentiation. These studies define a crucial early stage
of tumorigenesis and provide insight into its molecular basis.
Pre-neoplastic cells in patched mutant mice were first
described by Goodrich et al. as ‘regions of increased X-gal
staining on the surface of the cerebellum’ (Goodrich et al.,
1997). These cells have been suggested to represent either a
persistent external germinal layer or an early stage of
tumorigenesis (Corcoran and Scott, 2001; Goodrich et al.,
1997; Kim et al., 2003). The distinction is important: if these
cells are essentially normal GCPs (except for their persistence
into adulthood), their relevance
tumorigenesis may be limited. However, if they are partially
transformed and need only acquire a small number of
additional changes to become tumors, then studying their
properties may shed light on the earliest changes required in
medulloblastoma. Isolation of these cells has allowed us to
study their molecular and functional characteristics in detail.
Although pre-neoplastic cells resemble GCPs in terms of
location, lineage and hedgehog pathway activation, they differ
significantly from GCPs in terms of gene expression, including
loss of patched. In these respects, pre-neoplastic cells are much
more similar to tumor cells. Based on these observations, it is
likely that they represent an early stage of tumorigenesis.
In this context, it is worth noting that persistent EGL has
been observed in other mutant mice. For example, animals with
mutations in brain-derived neurotrophic factor (BDNF), matrix
metalloprotease 9 (MMP9), retinoid-related orphan receptor
alpha (RORα/staggerer) or the peroxisome assembly gene
PEX2 show delayed GCP migration and persistence of the
EGL (Borghesani et al., 2002; Faust, 2003; Messer and Hatch,
Development 132 (10)Research article
Table 1. Functions of differentially expressed genes
Gene symbolUnigene# Type of moleculePre:GCP Tumor:GCP
Secreted phosphoprotein 1 (osteopontin)
Unc5 homolog C
Collagen IV, alpha1
Paired box gene 6
CD47 (integrin-associated protein)
Protein tyrosine phosphatase, receptor type
Gap junction protein a1 (Connexin 43)
Platelet activating factor acetylhydrolase
Bcl2-associated athanogene 3
Activating transcription factor 3
Heat shock protein 105
DnaJ (Hsp40) homolog B1
DnaJ (Hsp40) homolog B10
FBJ osteosarcoma oncogene B
Forkhead box F2
Kruppel-like factor 4
Cysteine and glycine-rich protein 1
Zinc finger protein 288
Early growth response 2
Sry box containing gene 2
Neurogenic differentiation 1
Zinc finger protein of the cerebellum 3
Functions were assigned to genes based on information from Gene Ontology, Unigene, LocusLink and PubMed databases. Representative genes from the three
major functional groups are presented. Numbers refer to the fold change in gene expression between GCPs and pre-neoplastic cells (Pre:GCP), and between
GCPs and tumor cells (Tumor:GCP).
2435 Neoplastic stage of medulloblastomaDevelopment and disease
1984; Vaillant et al., 2003). Moreover, astrotactin 1 null mice
exhibit ectopic accumulations of GCPs that superficially
resemble the foci observed in patched heterozygotes (Adams
et al., 2002). However, none of these animals develop
medulloblastoma, suggesting that persistence of the EGL alone
is not sufficient for tumorigenesis. The fact that the ectopic
cells in patched mutant mice more closely resemble tumor cells
than GCPs – and occur in animals that develop tumors –
supports the use of the term ‘pre-neoplastic’ to describe these
Pre-neoplastic cells arise from proliferating granule
The fact that pre-neoplastic cells and tumor cells express
markers of the granule cell lineage is consistent with the notion
that they are derived from granule cell precursors. The same
has been postulated for human medulloblastomas, particularly
those with a desmoplastic appearance (Buhren et al., 2000;
Kadin et al., 1970; Miyata et al., 1998). Desmoplastic tumors
– which represent 20-30% of human medulloblastomas –
frequently harbor mutations in the Shh pathway, and have a
gene expression profile that resembles normal GCPs (Pomeroy
et al., 2002). Interestingly, recent studies have demonstrated
that some human medulloblastomas (including desmoplastic
tumors) express markers of neural stem cells (Hemmati et al.,
2003; Singh et al., 2003; Singh et al., 2004). This could mean
that they are derived from neural stem cells, or that they have
acquired stem cell markers as a consequence of transformation
(Oliver and Wechsler-Reya, 2004). Notably, few of the cells
that we isolate from the patched mutant mice express markers
of neural stem cells (nestin, CD133/prominin). Thus,
tumorigenesis in the patched mutant mice does not appear to
involve acquisition of a neural stem cell phenotype. Whether
these cells exhibit other properties of neural stem cells – such
as self-renewal or the capacity to differentiate into neurons and
glia – remains to be determined.
Loss of patched and activation of the hedgehog
pathway in pre-neoplastic cells
Our studies indicate that GCPs, pre-neoplastic cells and tumor
cells express elevated levels of hedgehog target genes and
proliferate in culture. In the case of GCPs, these responses
probably reflect exposure to Shh in vivo shortly before
isolation (Wechsler-Reya and Scott, 1999). In the case of pre-
neoplastic cells and tumor cells, de-repression of the hedgehog
pathway could result from loss of patched expression.
Fig. 10. Validation of
microarray data by
Cryosections of neonatal (P7)
cerebellum (A,D,G,J), pre-
neoplastic lesions (B,E,H,K)
and tumors (C,F,I,L) from
patched+/–mice were stained
with primary antibodies
specific for Zic3 (A-C), Pax6
(D-F), Necdin (G-I) or
Hsp105 (J-L) and peroxidase-
antibodies. Staining was
detected using the peroxidase
substrate DAB, which yields a
dark brown precipitate. Yellow
brackets mark the boundaries
of the EGL (panels A,D,G,J),
pre-neoplastic (PN) regions
(B,E,H,K) and tumor
(C,F,I,L). Expression of all
four proteins is detectable in
GCPs within the EGL.
Consistent with microarray
data, expression of Zic3
decreases significantly in pre-
neoplastic lesions and is
absent from tumors; Pax6 and
Necdin are restricted to
peripheral regions at the pre-
neoplastic stage and are
undetectable in tumors; and
Hsp105 expression increases
markedly at the pre-neoplastic
stage and decreases somewhat
Although some studies have suggested that wild-type patched
continues to be expressed in tumors (Romer et al., 2004;
Wetmore et al., 2000; Zurawel et al., 2000), others have
suggested that the wild-type patched locus in tumor cells may
be silenced (Berman et al., 2002). Our studies support the latter
view, demonstrating a striking loss of wild-type patched
expression at both the pre-neoplastic and tumor stages.
One critical feature of our studies is the use of primers that
distinguish between wild-type and mutant patched transcripts.
In most studies that have reported continued patched
expression in tumors from patched mutant mice, the data are
based on northern analysis or in situ hybridization using
probes that recognize both wild-type and mutant alleles.
Although the mutant allele is clearly expressed in tumors, it
is non-functional and would not be expected to contribute to
the behavior of tumor cells. Our studies are also distinct in
that we have separated pre-neoplastic and tumor cells from
normal tissue. This is important because several cell types in
the normal adult cerebellum express patched (Goodrich et al.,
1997; Traiffort et al., 1999), and these cells can contribute
significantly to RNA isolated from intact tumor tissue. By
FACS-sorting cells that express Math1-GFP, we have
eliminated these contaminating populations and analyzed
expression of patched specifically in pre-neoplastic cells and
The mechanism by which patched is lost in pre-neoplastic
cells and tumor cells remains unclear. Although studies of
medulloblastoma cell lines derived from patched+/–p53–/–mice
have demonstrated that the wild-type patched allele can be
silenced by methylation (Berman et al., 2002), we have found
no evidence for this in primary tumor cells from patched+/–
mice. Extensive sequencing of the four major CpG islands
within and upstream of the patched promoter revealed no
methylation in GCPs, pre-neoplastic cells or tumor cells (data
not shown). Although methylation (or some other chromatin
modification) could be present elsewhere in the patched gene,
it is also possible that loss of patched expression results from
mutations or deletions in the patched gene, or from loss of a
signaling molecule or transcription factor that regulates
patched expression. Mutational analysis of the patched locus,
and studies of the transcription factors bound to the promoter,
may help resolve this issue.
Regardless of the mechanism, our observation of decreased
wild-type patched expression in both pre-neoplastic cells and
tumor cells indicates that loss of patched is an early (and
perhaps initiating) event in tumorigenesis. The fact that only a
subset of pre-neoplastic cells develop into tumors implies that
other changes besides loss of patched are required for the
transition from the pre-neoplastic stage to more malignant
stages of medulloblastoma. Whether these changes result from
mutations or epigenetic events within pre-neoplastic cells
themselves, or whether they arise from changes in the
surrounding microenvironment, is an important question for
Identification of genes associated with
GCPs, pre-neoplastic cells and tumor cells resemble one
another in many ways. And yet, GCPs are present in all of
patched mutant mice (at the neonatal stage), pre-neoplastic
cells are present in over half of these animals (at 6 weeks), and
tumors occur in only 15-20%. Thus, these populations must
differ from one another. To identify intrinsic differences
between GCPs, pre-neoplastic cells and tumor cells, we
analyzed gene expression using microarrays. These studies
revealed that the three populations are remarkably similar
when compared with normal adult cerebellum, but show
significant differences when compared directly to one another.
Notably, because all three populations exhibit hedgehog
pathway activation and proliferation, differentially expressed
genes include few hedgehog targets or components of the cell
cycle machinery. Rather, major differences are detected
in genes associated with cell migration, survival and
Our approach differs from several recent studies of gene
expression analysis in medulloblastoma (Boon et al., 2003;
Kho et al., 2004; Lee et al., 2003; MacDonald et al., 2001;
Park et al., 2003; Pomeroy et al., 2002; Wechsler-Reya, 2003).
In most cases, these investigators compared medulloblastoma
with other brain tumors or with normal adult cerebellum. For
example, Pomeroy et al. found that medulloblastoma has a
distinct gene expression profile compared with other pediatric
brain tumors (Pomeroy et al., 2002). In particular,
mutations in the hedgehog pathway) have elevated expression
of hedgehog target genes such as Nmyc, patched, gli1 and
IGF2 (insulin-like growth factor 2). Similarly, Boon et al. used
serial analysis of gene expression (SAGE) to compare human
medulloblastoma and normal brain, and found increased
expression of hedgehog targets and cell cycle regulators such
as Nmyc and thymidylate synthase (Boon et al., 2003; Oliver
et al., 2003). These findings are consistent with the fact that
medulloblastoma cells are highly proliferative cells that
exhibit hedgehog pathway activation, whereas normal brain
consists primarily of post-mitotic neurons in which the
hedgehog pathway is inactive. In contrast to these studies, we
compared medulloblastoma cells with their cells of origin,
proliferating GCPs. This allowed us to control for
proliferation and hedgehog pathway activation and, instead, to
identify other genes that may play an important role in tumor
Similar to our studies, Lee et al. analyzed gene expression
in medulloblastoma and in neonatal cerebellum (Lee et
al., 2003). Both studies found that gene expression in
medulloblastoma was much more similar to neonatal
cerebellum than to adult cerebellum. Yet there were also key
differences between these studies. In particular, Lee et al. found
that medulloblastoma is characterized by increased expression
of genes associated with the granule cell lineage, hedgehog
pathway activation and proliferation
hexokinase, Nmyc, cyclin D1, sfrp1), whereas we did not detect
elevated expression of these genes. One important distinction
between the two studies is the use of intact tissues versus
dissociated cells. Lee et al. compared intact tumor tissue
(which consists largely of GCP-like cells) and intact neonatal
cerebellum (which contains not only GCPs but also significant
numbers of post-mitotic granule cells and other cell types). By
contrast, we compared purified populations of GCPs, pre-
neoplastic cells and tumor cells, all of which are highly
enriched for proliferating GCP-like cells. As a result, genes
associated with cell lineage and degree of hedgehog pathway
activity were not differentially expressed in our screen.
(which often harbor
Development 132 (10)Research article
2437 Neoplastic stage of medulloblastomaDevelopment and disease
Dysregulation of migration, differentiation and
apoptosis in medulloblastoma
Our study is unique in that we compared gene expression in
similar populations of cells at three different stages of tumor
progression. This allowed us to identify genes that distinguish
these stages. Interestingly, many of these genes were regulators
of migration, survival and differentiation, processes that have
been studied in the context of normal granule cell development
but were not previously known to play a role in
medulloblastoma. Most prominent among these genes were
regulators of cell migration. These included transcription
factors (Pax6), surface receptors (Unc5h3), secreted proteins
(PAF acetylhydrolase) and ECM molecules (Collagen) that
have been implicated in granule cell migration (Engelkamp et
al., 1999; Fishman and Hatten, 1993; Przyborski et al., 1998;
Tokuoka et al., 2003). Notably, the majority of the genes we
identified have been found to promote migration, and were
downregulated in pre-neoplastic cells. Decreased expression of
these genes during the early stages of tumorigenesis is
interesting because pre-neoplastic cells clearly exhibit aberrant
migration: whereas GCPs migrate inward during normal
development, pre-neoplastic cells (and tumor cells) remain
stuck on the surface of the cerebellum. A similar location has
been noted for human medulloblastomas, which often spread
through the meninges but rarely invade the inner layers of the
cerebellum (Koeller and Rushing, 2003).
It has long been unclear how failure to migrate inward is
related to tumorigenesis. One possibility is that pre-neoplastic
cells and tumor cells are unable to migrate because they are
proliferating or locked in a precursor state. Alternatively, the
inability to migrate could be an intrinsic defect that plays an
essential role in tumorigenesis. For example, by losing the
ability to migrate, cells may remain trapped in an environment
(e.g. close to the pial surface) that facilitates their continued
growth and survival. Our finding that genes involved in
migration are downregulated in pre-neoplastic cells strongly
supports the notion of an intrinsic defect. However, further
experiments will be necessary to determine whether changes
in migration-associated genes are necessary for the
development of medulloblastoma.
Genes that regulate differentiation may also be crucial in
determining the course of tumorigenesis. Importantly, we
found that pre-neoplastic cells have reduced expression of
genes associated with granule cell differentiation (e.g. Zic3 and
Neurod1). This is consistent with the fact that these cells
continue to proliferate and retain expression of GCP markers,
whereas normal GCPs can exit the cell cycle and differentiate
into mature granule neurons. Whether the inability to
differentiate is a cause or a consequence of transformation
remains to be determined, but the identification of specific
transcription factors that may regulate this process will allow
us to test the significance of differentiation directly. Further
studies perturbing the expression of these factors in vivo are
Finally, our data suggest that altered stress responses may
contribute to tumor formation in patched mutant mice. Heat-
shock proteins, co-chaperones (Hsp60, Hsp105, Dnajb1,
Dnajb10 and Bag3) and the transcription factor Atf3 all
function to protect cells from stress-induced apoptosis and
necrosis (Hatayama et al., 2001; Nakagomi et al., 2003;
Takayama et al., 2003). The increased expression of these
genes in pre-neoplastic cells and tumor cells may reflect an
increased ability to survive under stress conditions, which may
be crucial for the early stages of tumorigenesis. In fact, several
lines of evidence suggest that dysregulation of apoptosis or
stress responses may be important for medulloblastoma
formation. For example, crossing patched mutant mice with
homozygous p53 knockout mice leads to a dramatic increase
in tumor incidence: whereas only 14-20% of patched
heterozygotes develop medulloblastoma, more than 95% of
patched+/–/p53–/–mice develop tumors (Wetmore et al., 2001).
Similarly, animals that lack both p53 and PARP1, an enzyme
that promotes cell death in response to DNA damage, develop
medulloblastoma (Tong et al., 2003). Notably, no mutations in
p53 have been found in tumors from patched heterozygotes
(Wetmore et al., 2001), suggesting that in these animals some
other element of the apoptotic machinery may need to be
inactivated for tumors to form. The genes we have found to
be upregulated in pre-neoplastic cells may provide some
important clues to the mechanisms by which apoptosis is
subverted in medulloblastoma.
The identification of a pre-neoplastic stage in murine
medulloblastoma raises the possibility that a similar stage
exists in the human disease. If so, the genes we have identified
as markers of pre-neoplastic cells may be useful for early
detection of medulloblastoma, particularly in people with an
inherited susceptibility to the disease (Gorlin, 1987; Hamilton
et al., 1995). Detection of medulloblastoma at early stages may
increase the effectiveness of conventional medulloblastoma
therapy. In addition, if the genes we have identified play a
causal role in tumor progression, they may serve as valuable
targets for therapy. Recent studies suggest that pharmacologic
antagonists of the hedgehog pathway may be effective at
inhibiting growth of medulloblastoma cells in vitro and in vivo
(Berman et al., 2002; Romer et al., 2004). Therefore,
identification of other pathways that are crucial for
tumorigenesis may open up new avenues for treatment of this
We thank Holly Dressman, Laura Reid, and the Duke Microarray
Core Facility for microarray processing, Mike Cook and Lynn
Martinek for flow cytometric analysis, and Thomas Cummings for
interpretation of histological staining. We are also grateful to Wieland
Huttner and Denis Corbeil for anti-prominin/CD133 antibodies, to
Jane Johnson for Math1-GFP mice, to Audra Carroll for helpful
discussions, and to Terry Van Dyke, Phil Beachy and Chris Counter
for critical review of the manuscript. T.G.O. is a National Science
Foundation graduate student fellow and R.J.W.-R. is a Kimmel
Foundation Scholar. This research was supported by a McDonnell
Foundation 21st Century Award and by funds from the Children’s
Brain Tumor Foundation.
Supplementary material for this article is available at
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