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First published online 20 April 2005
doi: 10.1242/dev.01793

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Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma

¹ Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
² Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA
³ Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC 27710, USA

View larger version (50K): [in a new window]   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 ß-galactosidase gene. At earlier ages, >50% of patched mutant mice have ectopic ß-galactosidase-expressing cells (arrowheads in B). Background X-gal staining was not detected in the cerebellum of adult wild-type mice (A).

View larger version (73K): [in a new window]   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.

View larger version (91K): [in a new window]   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.

View larger version (60K): [in a new window]   Fig. 4. Co-expression of Math1 and ß-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.

View larger version (9K): [in a new window]   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 ß-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.

View larger version (19K): [in a new window]   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.

View larger version (16K): [in a new window]   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 ß-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.

View larger version (15K): [in a new window]   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.

View larger version (60K): [in a new window]   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 differentially expressed. 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.

View larger version (145K): [in a new window]   Fig. 10. Validation of microarray data by immunohistochemistry. 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-conjugated secondary 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 in tumors.

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