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Dual and opposing roles of primary cilia in medulloblastoma development
© 2009 Nature America, Inc. All rights reserved.
Young-Goo Han1, Hong Joo Kim2, Andrzej A Dlugosz3, David W Ellison4, Richard J Gilbertson5 & Arturo Alvarez-Buylla1 Recent work has shown that primary cilia are essential for Hedgehog (Hh) signaling during mammalian development1–9. It is also known that aberrant Hh signaling can lead to cancer10, but the role of primary cilia in oncogenesis is not known. Cerebellar granule neuron precursors (GNPs) can give rise to medulloblastomas, the most common malignant brain tumor in children11. The primary cilium and Hh signaling are required for GNP proliferation8,12–15. We asked whether primary cilia in GNPs have a role in medulloblastoma growth in mice. Genetic ablation of primary cilia blocked medulloblastoma formation when this tumor was driven by a constitutively active Smoothened protein (Smo), an upstream activator of Hh signaling. In contrast, removal of cilia was required for medulloblastoma growth by a constitutively active glioma-associated oncogene family zinc finger-2 (GLI2), a downstream transcription factor. Thus, primary cilia are either required for or inhibit medulloblastoma formation, depending on the initiating oncogenic event. Remarkably, the presence or absence of cilia was associated with specific variants of human medulloblastomas; primary cilia were found in medulloblastomas with activation in HH or WNT signaling but not in most medulloblastomas in other distinct molecular subgroups. Primary cilia could serve as a diagnostic tool and provide new insights into the mechanism of tumorigenesis. Abnormal activation of Hh signaling, through loss of the Hh receptor Patched1 (Ptch1) or activation of Smo, induces medulloblastomas in mice16–21. To induce medulloblastoma, we expressed constitutively active Smo (SmoM2) in GNPs using a human glial fibrillary acidic protein promoter-driven Cre (GFAPCre)20. By postnatal day 10 (P10), GFAPCre;SmoM2fl/+ mice developed medulloblastoma (n = 7) (Fig. 1a). In these tumors, SmoM2 fused with yellow fluorescent protein (YFP) localized to primary cilia that are associated with the basal body (Fig. 1b). To investigate whether SmoM2-driven medulloblastoma formation requires primary cilia, we removed primary cilia from GNPs expressing SmoM2, using a conditional allele of Kif3a, which encodes a subunit of the kinesin-II motor essential for ciliogenesis22–24. The removal of Kif3a and the consequent loss of cilia completely blocked tumorigenesis (n = 7) (Fig. 1a).
The cerebella of the GFAPCre;SmoM2fl/+;Kif3afl/fl mice resembled those of GFAPCre;Kif3afl/fl mice (Fig. 1a), which fail to expand GNPs8,12. Loss of Ift88, encoding intraflagellar transport 88 homolog, another gene essential for ciliogenesis25,26, in GFAPCre;SmoM2fl/+; Ift88fl/fl mice also blocked tumorigenesis driven by SmoM2 (Supplementary Fig. 1a). At embryonic day 16 (E16), when the number of GNPs is not affected by removing cilia8, the external granule cell layer (EGL) was already expanded, with many proliferating cells in GFAPCre;SmoM2fl/+ mice but not in GFAPCre;SmoM2fl/+;Kif3afl/fl mice (Fig. 1c,d), suggesting that SmoM2 requires Kif3a to initiate the aberrant GNP expansion and medulloblastoma. A similar requirement of cilia for SmoM2-driven expansion of GNPs has been observed in the hippocampal dentate gyrus4. Activated Smo converts Gli2 into a transcriptional activator and inhibits the formation of Gli3 repressors that form constitutively in the absence of Hh signaling27. We hypothesized that constitutively active Gli2 could induce medulloblastomas in the absence of primary cilia. To test this hypothesis, we used CLEG2 transgenic mice that, upon Cre-mediated recombination, express a constitutively active human GLI2 that lacks the N-terminal repressor domain (GLI2∆N)28,29. Unexpectedly, none of the GFAPCre;CLEG2fl/+ mice (n = 14) developed medulloblastoma (Fig. 2), albeit two had another type of tumor (detailed below). To our surprise, unlike in GFAPCre;CLEG2fl/+ mice, removal of primary cilia in GFAPCre;CLEG2fl/+;Kif3afl/fl mice induced medulloblastomas between P11 and P30 (n = 11) (Fig. 2). Tumors in GFAPCre;CLEG2fl/+;Kif3afl/fl mice contained two types of cells frequently segregated into distinct zones: cells with darkly stained nuclei and lightly stained cytoplasm (type 1) and cells with large nuclei and highly eosinophilic cytoplasm (type 2) (Fig. 2b,c). Both cell types were actively proliferating as determined by BrdU incorporation (Fig. 2f) and expressed Gli1, suggesting active Hh signaling (Supplementary Fig. 2). The type 1 tumor cells were indistinguishable from those in SmoM2-driven medulloblastoma17,18,20 and were reminiscent of classic medulloblastoma cells: small round cells with high nuclear-to-cytoplasmic ratio. Type 1 cells showed immunohistological characteristics of medulloblastoma similar to those in SmoM2 tumor: neuronal (βIII tubulin), glial (GFAP and oligodendrocyte transcription factor-2 (Olig2)), GNP (paired box gene-6 (Pax6)), and granule neuron (zinc finger protein of the cerebellum-1
1Department
of Neurological Surgery and The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research and 2Gladstone Institute of Neurological Disease, University of California–San Francisco (UCSF), California, USA. 3Department of Dermatology and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan, USA. 4Department of Pathology and 5Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA. Correspondence should be addressed to A.A.-B. (
[email protected]). Received 5 February; accepted 28 July; published online 23 August 2009; doi:10.1038/nm.2020
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Figure 1 Kif3a is required for SmoM2-driven medulloblastoma formation. (a) Hematoxylin-stained sagittal sections of control and mutant cerebella at P10. (b) Immunofluorescence showing the localization of SmoM2-YFP. Antibody to GFP shows SmoM2-YFP is highly enriched in primary cilia (green, arrow) associated with the basal body (γ-tubulin–specific staining, shown in red, arrowhead) in the tumors in GFAPCre;SmoM2fl/+;Kif3afl/+ mice. In GFAPCre;SmoM2fl/+;Kif3afl/fl mice, only the basal body is present (arrowhead). (c,d) BrdU incorporation (1-h survival) at E16. Sagittal sections of cerebella are stained with antibody to BrdU (green). Nuclei are stained with DAPI (blue) (c). BrdU+ cells in the EGL are counted by using ImageJ program. Error bars are s.e.m. (d). *P < 0.05. Scale bars: 0.5 mm (a), 5 µm (b) and 100 µm (c).
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(Zic1)) markers (Supplementary Fig. 3). GFAPCre;CLEG2fl/+; Kif3afl/fl and GFAPCre;SmoM2fl/+ mice also had very similar gene expression profiles (Fig. 2h), including upregulation of Hh-responsive genes characteristic of medulloblastoma cells (Gli1, Gli2, Ptch1, Ptch2, Mycn (encoding N-Myc), Ccnd2, (encoding cyclin D2) Atoh1 (encoding atonal homolog-1) and Bmi1 (encoding Bmi1 polycomb ring finger oncoprotein)). The expression of Gli3 and Wif1 (encoding Wnt inhibitory factor-1) differed between the two tumors, probably owing to the presence of type 2 tumor cells in GFAPCre;CLEG2fl/+;Kif3afl/fl mice; type 2 tumor cells downregulated Gli3 and upregulated Wif1 (Fig. 2h and detailed below). Type 2 tumors lacked immunohistological
characteristics of medulloblastoma markers (Supplementary Fig. 3), but expressed Sox2 (sex determining region Y-box 2) (data not shown), suggesting that these are not medulloblastomas. We also found type 2 tumors outside of the cerebellum, suggesting that these cells have a different origin (Supplementary Fig. 4a). Both tumor types lacked primary cilia (Supplementary Fig. 5). Similar to loss of Kif3a, loss of Ift88 promoted tumor formation containing both type 1 and type 2 tumor cells in GFAPCre;CLEG2fl/+;Ift88fl/fl mice (Supplementary Fig. 1b). Later in life, GFAPCre;CLEG2fl/+ mice developed only type 2 tumors, which we found throughout central nervous system (Supplementary Fig. 4b), and they lived significantly longer than GFAPCre; CLEG2fl/+;Kif3afl/fl mice (P < 0.0001, n = 19) (Fig. 2g). These tumors expressed high levels of Hh-responsive genes (Gli1, Gli2, Ptch1, Ptch2, Mycn and Ccnd2) and Wnt-responsive genes (Axin2 and Wif1) but not genes associated with GNPs and medulloblastoma (Atoh1 (ref. 19) and Bmi1 (ref. 30)) (Fig. 2h). Type 2 tumors in GFAPCre;CLEG2fl/+ mice had primary cilia, excluding the possibility that they may have originated from cells that spontaneously lost primary cilia (Supplementary Fig. 5). Thus, loss of cilia was required for medulloblastoma development driven
Figure 2 Kif3a suppresses GLI2∆N-driven fl/+ GFAP::Cre;CLEG2 ; fl/+ fl/fl medulloblastoma formation. (a–c) H&E-stained GFAP::Cre;CLEG2 kif3a 1 2 3 sagittal sections of control and mutant cerebella 240 Gli3 140 at P23. (a) GFAPCre;CLEG2fl/+ mice have * ectopic clusters of cells (arrows) without tumor 100 Gli3R formation. (b) All GFAPCre;CLEG2fl/+;Kif3afl/fl 70 1. WT mice develop medulloblastomas between P11 fl/+ 2. GFAP::Cre;CLEG2 and P30. A green dotted line in b demarcates fl/+ fl/fl Type 2 Type 1 3. GFAP::Cre;CLEG2 ;Kif3a domains of the tumors containing type 1 100 and type 2 tumor cells (see text). (c) High fl/+ fl/fl GFAP::Cre;CLEG2 ;Kif3a 80 magnification of red box in b, showing the GFAP::Cre;CLEG2 fl/+;Gli3Xt/+ 60 two domains with different cell types. fl/+ GFAP::Cre;CLEG2 (d) Western blot analysis of Gli3 40 proteins isolated from P23 cerebella in 20 GFAPCre;CLEG2fl/+; Kif3afl/fl mice compared P23 with wild-type (WT) and GFAPCre;CLEG2fl/+ 50 100 150 Time (d) mice. Asterisk indicates a nonspecific band, * which serves as a loading control. This banding Gli1 Gli2 Gli3 Ptch1 16 Ptch2 Mycn Ccnd2 Atoh1 * pattern is similar to a previous report, which 14 Bmi1 Axin2 Wif1 showed that this Gli3 antibody detects specific 12 Gli3 bands that are absent in Gli3 mutants and 10 * * * * * nonspecific bands38. (e,f) BrdU incorporation 8 * * * * * * * * 6 (1-h survival, green) in GFAPCre;CLEG2fl/+ * * * * ** * * * * * 4 mice and GFAPCre;CLEG2fl/+;Kif3afl/fl mice at * * * 2 * P23. Ectopic clusters in GFAPCre;CLEG2fl/+ * ** * 0 mice are not proliferating (arrows). Nuclei –2 are stained with DAPI (red). (g) Survival * * * GFAP::Cre; GFAP::Cre; GFAP::Cre; GFAP::Cre; analysis of GFAPCre;CLEG2fl/+;Kif3afl/fl fl/+ fl/+ fl/fl fl/+ Xt/+ fl/+ CLEG2 CLEG2 ;Kif3a CLEG2 ;Gli3 SmoM2 mice,GFAPCre;CLEG2fl/+;Gli3Xt/+ mice and GFAPCre;CLEG2fl/+ mice. (h) The expression levels of Hh and Wnt target genes in tumors, determined by quantitative RT-PCR analyses. Error bars represent s.e.m. *P < 0.05. Scale bars: 0.5 mm (a,b,e,f) and 10 µm (c).
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by GLI2∆N but not for type 2 tumors; yet, loss of cilia accelerated the development of the type 2 tumor (Fig. 2a–c,g). The above results suggest that loss of primary cilia is required for GLI2∆N-driven medulloblastoma development, which is in striking contrast to their requirement for SmoM2-driven tumorigenesis. One possible explanation for these opposite roles is that primary cilia are required not only for Smo function but also for Gli3 repressor formation2,6,7,9. Consistently, Gli3 repressor level was markedly lower in GFAPCre;CLEG2fl/+;Kif3afl/fl mice than in wild-type or GFAPCre;CLEG2fl/+ mice (Fig. 2d). Gli3 expression was also significantly lower in type 2 tumors in GFAPCre;CLEG2fl/+ mice (Fig. 2h), suggesting that Gli3 might also have an inhibitory role in these tumors. Because homozygosity for the null mutation of Gli3 gene (Gli3 extra toes (Xt)) is embryonic lethal, we removed one copy of Gli3 in mice expressing GLI2∆N (GFAPCre;CLEG2fl/+;Gli3Xt/+) to test whether Gli3 has an inhibitory effect on GLI2∆N-driven tumorigenesis. GFAPCre;CLEG2fl/+;Gli3Xt/+ mice developed type 2 tumors and died earlier in life than GFAPCre;CLEG2fl/+ mice (P < 0.02, n = 18) (Fig. 2g and Supplementary Fig. 6). Thus, loss of one copy of Gli3 induces earlier development of the type 2 tumors in the presence of primary cilia, supporting our inference that Gli3 repressors may mediate the inhibitory function of primary cilia in tumorigenesis driven by GLI2∆N. Of note, GFAPCre;CLEG2fl/+;Gli3Xt/+ mice did not develop medulloblastoma, possibly owing to the repressor activity of one copy of wild-type Gli3. Loss of primary cilia might also interfere with other signaling pathways that contribute to medulloblastoma development. Whereas SMOM2 mutations have been observed in one case of human medulloblastoma31, activating mutations in GLI transcription factors have not been described in medulloblastoma. Thus, our mouse models reveal key roles of primary cilia in tumorigenesis but might not reflect on the behavior of this organelle in human cases. To determine whether our observations in mice are relevant to human tumors, we analyzed 38 human medulloblastomas for the presence of cilia. The presence of primary cilia was significantly associated with desmoplastic medulloblastoma (five of six tumors were ciliated; P < 0.02); whereas the absence of cilia was significantly associated with anaplastic medulloblastoma (only one of nine tumors was ciliated; P < 0.05) (Fig. 3a and Table 1). Twenty-four of these samples were analyzed previously for gene expression profiling32. Notably,
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Figure 3 Primary cilia are present in a subset of human medulloblastomas. (a) Immunofluorescence showing the presence or absence of primary cilia in medulloblastomas. Primary cilia are stained with an antibody to acetylated tubulin (green, arrows), and basal bodies are stained with an antibody to γ-tubulin (red, arrowheads). An example of nonciliated tumor is shown for anaplastic medulloblastoma. The numbers of samples examined and of ciliated samples are given under each type of medulloblastoma. Scale bar, 5 µm. (b) Model summarizing the dual roles of primary cilia in SmoM2-driven and GLI2∆N-driven tumorigenesis. SmoM2 is insensitive to inhibition by Ptch1 and constitutively localizes to the cilia, where it activates Gli2 and inhibits Gli3 repressor (Gli3R) formation, leading to medulloblastoma (top left). Without the cilia, SmoM2 cannot activate downstream pathways; the cerebellum remains small as in ciliary mutants8 and no tumors develop (bottom left). GLI2∆N is constitutively active and independent of primary cilia, yet mice expressing this mutant protein do not develop medulloblastoma, possibly owing to the presence of Gli3 repressor. However, these mice develop type 2 tumors later in life (top right). In the absence of cilia, GLI2∆N induces medulloblastoma and type 2 tumors earlier in life. This may be the result of the elimination of Gli3 repressor when cilia are removed (bottom right).
in these 24 samples, we identified cilia almost exclusively in tumors that had activation in either HH or WNT signaling (P < 0.0001, Table 1). Two of the four tumors with active HH signaling had inactivating mutations in PTCH1 (ref. 32), which functions upstream of SMO and primary cilia5. Our results suggest that primary cilia can be either permissive or inhibitory for tumor formation, depending on the underlying oncogenic events (Fig. 3b). We found that some subsets of human medulloblastomas have primary cilia, whereas others do not. Our mouse models predict that medulloblastomas with activating mutations in HH signaling components downstream of the cilia will grow only when the cilia are not present. Notably, all of the observed human medulloblastomas with high HH signaling activity had primary cilia. This suggests that medulloblastomas with high HH signaling in humans largely result from mutations upstream of the primary Table 1 Primary cilia are present almost exclusively in medulloblastomas with either HH or WNT signaling activation
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Classic Classic Classic Classic Classic Classic Classic
WT WT WT WT WT WT WT
WT WT WT WT WT WT WT
Yes Yes Yes Yes
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Desmoplastic Desmoplastic Desmoplastic Anaplastic
Yes Yes Yes Yes/equivc
B B B B
Classic Classic Classic Classic
Yes Equiv No No No No No
A A A A A A A
Equiv No No/equiv No No No No No No
Concurrent deletion of chromosome 17p and gain of 17q
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subgroups are determined (A to E) on the basis of gene expression profiles32. bHistopathological type of medulloblastomas. cSamples for which it was hard to determine the presence of cilia owing to suboptimal staining are labeled as ‘equiv’.
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Letters cilia, such as mutations in PTCH1. Oncogenic mutations in downstream molecules, including GLI transcription factors, which would require loss of repressor activity mediated by the cilia, are rare11. A recent study identified rare amplifications of GLI1 and GLI2 genes in medulloblastomas33, but it is not known whether these tumors are ciliated. Analyses of more medulloblastomas both for oncogenic mutations and for the presence or absence of primary cilia will be necessary to delineate the role of cilia in human medulloblastoma. Of note, all of the observed human medulloblastomas with high WNT signaling had primary cilia, and all of these tumors had mutations in CTNNB1, which encodes β-catenin. It is possible that primary cilia are also involved in the WNT signaling pathway34–36 or that oncogenic WNT activation requires concomitant signaling through primary cilia to induce medulloblastoma. Our observations suggest that the presence of primary cilia is fundamental for tumor diagnosis and possibly for the development of new treatments. HH signaling mutations found in medulloblastomas, loss of PTCH1 and activation of SMO all act upstream of primary cilia2,4–9. Ciliated medulloblastomas showing high HH signaling would be susceptible to treatments that target primary cilia. The loss of cilia in subgroups of medulloblastomas suggests that tumor repressor functions mediated through primary cilia might serve as therapeutic targets to inhibit tumor growth. GLI3 is a strong candidate, but primary cilia could have multiple repressor activities. Understanding the role of primary cilia in normal and oncogenic signaling may lead to better understanding of tumor development and treatment not only in medulloblastomas but also in other tumors37. Methods Methods and any associated references are available in the online version of the paper at http://www.nature.com/naturemedicine/. Note: Supplementary information is available on the Nature Medicine website. Acknowledgments We thank L.S. Goldstein at the University of California–San Diego for providing us with Kif3afl/fl mice; D. Rowitch at UCSF for SmoM2fl/+ mice; B. Yoder at the University of Alabama–Birmingham for Ift88fl/fl mice; C. Cowdrey and the Neurological Surgery Tissue Bank at UCSF for human medulloblastoma samples; R. Segal and C. Stiles at Harvard University for Zic–specific and Olig2–specific antibodies; and A. Ruiz i Altaba at University of Geneva Medical School for Gli1 cDNA. We thank S. Wong, J. Reiter, D. Cano and S. Cervantes-Roldan for sharing unpublished data. We thank S. Vandenberg for helping with assessing the tumor types; J. Morris, K. Blaschke and M. Sachs for helping with quantitative RT-PCR; R. Romero for technical assistance; and D. Rowitch, J. Reiter, S Wong, R. Ihrie, S. Nader and T. Nguyen for comments on the manuscript. Y.-G.H. was, in part, supported by Mark Linder/American Brain Tumor Association Fellowship. The work was supported by grants from the US National Institutes of Health (NS28478 and HD32116), John G Bowes Research Fund and a grant from the Goldhirsh Foundation to A. A.-B. Confocal microscopy at Diabetes & Endocrinology Research Center. Microscopy and Imaging Core was supported by an US National Institutes of Health grant P30 DK063720. AUTHOR CONTRIBUTIONS Y.-G.H. designed and performed most experiments. H.J.K. performed western blot analysis. A.A.D. provided CLEG2 mice. D.W.E. and R.J.G. provided human medulloblastoma tissue microarrays that were analyzed previously for gene expression profiling. A.A.-B. supervised the project. Y.-G.H. and A.A.-B. wrote the manuscript. All authors commented on the manscript. Published online at http://www.nature.com/naturemedicine/. Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/. 1. Corbit, K.C. et al. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021 (2005).
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2. Haycraft, C.J. et al. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet. 1, e53 (2005). 3. Rohatgi, R., Milenkovic, L. & Scott, M.P. Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372–376 (2007). 4. Han, Y.G. et al. Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells. Nat. Neurosci. 11, 277–284 (2008). 5. Huangfu, D. et al. Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426, 83–87 (2003). 6. Liu, A., Wang, B. & Niswander, L.A. Mouse intraflagellar transport proteins regulate both the activator and repressor functions of Gli transcription factors. Development 132, 3103–3111 (2005). 7. May, S.R. et al. Loss of the retrograde motor for IFT disrupts localization of Smo to cilia and prevents the expression of both activator and repressor functions of Gli. Dev. Biol. 287, 378–389 (2005). 8. Spassky, N. et al. Primary cilia are required for cerebellar development and Shhdependent expansion of progenitor pool. Dev. Biol. 317, 246–259 (2008). 9. Huangfu, D. & Anderson, K.V. Cilia and Hedgehog responsiveness in the mouse. Proc. Natl. Acad. Sci. USA 102, 11325–11330 (2005). 10. Varjosalo, M. & Taipale, J. Hedgehog: functions and mechanisms. Genes Dev. 22, 2454–2472 (2008). 11. Gilbertson, R.J. & Ellison, D.W. The origins of medulloblastoma subtypes. Annu. Rev. Pathol. 3, 341–365 (2008). 12. Chizhikov, V.V. et al. Cilia proteins control cerebellar morphogenesis by promoting expansion of the granule progenitor pool. J. Neurosci. 27, 9780–9789 (2007). 13. Dahmane, N. & Ruiz i Altaba, A. Sonic hedgehog regulates the growth and patterning of the cerebellum. Development 126, 3089–3100 (1999). 14. Wallace, V.A. Purkinje-cell–derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum. Curr. Biol. 9, 445–448 (1999). 15. Wechsler-Reya, R.J. & Scott, M.P. Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 22, 103–114 (1999). 16. Goodrich, L.V., Milenkovic, L., Higgins, K.M. & Scott, M.P. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277, 1109–1113 (1997). 17. Hallahan, A.R. et al. The SmoA1 mouse model reveals that notch signaling is critical for the growth and survival of sonic hedgehog-induced medulloblastomas. Cancer Res. 64, 7794–7800 (2004). 18. Mao, J. et al. A novel somatic mouse model to survey tumorigenic potential applied to the Hedgehog pathway. Cancer Res. 66, 10171–10178 (2006). 19. Oliver, T.G. et al. Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma. Development 132, 2425–2439 (2005). 20. Schüller, U. et al. Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma. Cancer Cell 14, 123–134 (2008). 21. Yang, Z.J. et al. Medulloblastoma can be initiated by deletion of Patched in lineagerestricted progenitors or stem cells. Cancer Cell 14, 135–145 (2008). 22. Rosenbaum, J.L. & Witman, G.B. Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3, 813–825 (2002). 23. Marszalek, J.R. et al. Genetic evidence for selective transport of opsin and arrestin by kinesin-II in mammalian photoreceptors. Cell 102, 175–187 (2000). 24. Marszalek, J.R., Ruiz-Lozano, P., Roberts, E., Chien, K.R. & Goldstein, L.S. Situs inversus and embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of kinesin-II. Proc. Natl. Acad. Sci. USA 96, 5043–5048 (1999). 25. Haycraft, C.J. et al. Intraflagellar transport is essential for endochondral bone formation. Development 134, 307–316 (2007). 26. Murcia, N.S. et al. The Oak Ridge polycystic kidney (orpk) disease gene is required for left-right axis determination. Development 127, 2347–2355 (2000). 27. Huangfu, D. & Anderson, K.V. Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways from Drosophila to vertebrates. Development 133, 3–14 (2006). 28. Pasca di Magliano, M. et al. Hedgehog/Ras interactions regulate early stages of pancreatic cancer. Genes Dev. 20, 3161–3173 (2006). 29. Roessler, E. et al. A previously unidentified amino-terminal domain regulates transcriptional activity of wild-type and disease-associated human GLI2. Hum. Mol. Genet. 14, 2181–2188 (2005). 30. Leung, C. et al. Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas. Nature 428, 337–341 (2004). 31. Lam, C.W. et al. A frequent activated smoothened mutation in sporadic basal cell carcinomas. Oncogene 18, 833–836 (1999). 32. Thompson, M.C. et al. Genomics identifies medulloblastoma subgroups that are enriched for specific genetic alterations. J. Clin. Oncol. 24, 1924–1931 (2006). 33. Northcott, P.A. et al. Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma. Nat. Genet. 41, 465–472 (2009). 34. Corbit, K.C. et al. Kif3a constrains β-catenin–dependent Wnt signalling through dual ciliary and non-ciliary mechanisms. Nat. Cell Biol. 10, 70–76 (2008). 35. Gerdes, J.M. et al. Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response. Nat. Genet. 39, 1350–1360 (2007). 36. Simons, M. et al. Inversin, the gene product mutated in nephronophthisis type II, functions as a molecular switch between Wnt signaling pathways. Nat. Genet. 37, 537–543 (2005). 37. Wong, S.Y. et al. Nat. Med. advance online publication, doi:10.1038/nm.2011 (23 August 2009). 38. Martinelli, D.C. & Fan, C.M. Gas1 extends the range of Hedgehog action by facilitating its signaling. Genes Dev. 21, 1231–1243 (2007).
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ONLINE METHODS Mice. All mouse procedures were approved by the UCSF Institutional Animal Care and Use Committee. Genetically modified mice, GFAPCre39 (obtained from Jackosn Laboratory), Kif3afl/fl (ref. 23), SmoM2-YFPfl/fl (ref. 18),Ift88fl/fl (ref. 25) and CLEG2 (ref. 28) were described previously.
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Tissue preparation and staining. We injected mice with BrdU (50 mg per kg body weight) and perfused them 1h later with 4% paraformaldehyde. We postfixed the brains overnight at 4°C. For frozen sections, we cryoprotected brains in 30% sucrose, embedded in them in optimal cutting temperature medium (Sakura) and cut them into 10-µm slices. For polyethylene glycol (PEG) sections, we dehydrated the brains in an ethanol series, embedded them in a 2:1 mixture of PEG 1000 and PEG 1500 and cut them into 8-µm slices. For immunostaining, we incubated the sections with primary antibodies overnight at 4 °C followed by incubation with secondary antibodies at room temperature (20–25 °C) for 2 h. We took images with an Olympus AX70 microscope or Leica SP2 or SP5 confocal microscopes and processed them with Adobe Photoshop. Antibodies and reagents. We used the following primary antibodies: rat antibody to BrdU (1 in 1,000, ab209, Abcam), mouse antibodies to acetylated tubulin (1 in 1,000, T6793, Sigma), to GFAP (1 in 1,000, MAB3402, Chemicon) and to Tuj1 (1 in 500, MMS-435PCovance), rabbit antibodies to γ-tubulin (1 in 1,000, T5192, Sigma), to Zic (recognizes Zic1 and other Zic proteins, gift from R. Segal, 1 in 3,000), to Olig2 (DF308, gift from C. Stiles, 1 in 20,000) and to pericentrin (1 in 1,000, ab4448, Abcam), goat antibody to Pax6 (1 in 50, sc7750, Santa Cruz) and chicken antibody to GFP (1 in 500, GFP-1020, Aves Labs). We visualized SmoM2-YFP with GFP-specific antibody. We used Alexa Fluor–conjugated secondary antibodies (1 in 400, Molecular Probes), and we used DAPI (200 ng ml–1, D9542, Sigma) for counterstaining. We also used standard H&E staining. Gli1 in situ hybridization. We used a standard in situ protocol and an antisense riboprobe from Gli1 complementary DNA (gift from A. Ruiz i Altaba). Western blotting. We lysed whole cerebella from P23 mice in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% SDS, Protease inhibitors (Roche, 11 697 498 001)). We removed pellets after centrifugation. We
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resolved lysates by 7% SDS-PAGE. We used rabbit Gli3-specific antibody (1 in 500, Santa Cruz, H280) for immunoblotting. This Gli3 antibody detects specific Gli3 bands that are absent in Gli3 mutants and nonspecific bands38. Quantitative reverse transcription PCR. We isolated total RNA from each tumor of an individual mouse (n = 3 per group) with the RNeasy Mini Kit (Qiagen) and reverse transcribed it with Superscript III reverse transcriptase (Invitrogen). We performed quantitative PCR on an Applied Biosystems 7900 quantitative PCR instrument using SYBR green (Applied Biosystems, 4309155). We normalized transcript levels to the expression levels of Gapdh (encoding glyceraldehyde-3-phosphate dehydrogenase) and measured them relative to those in wild-type cerebellum. Primer sequences are listed in Supplementary Table 1. Human medulloblastoma samples. We prepared sections from formalin-fixed paraffin-embedded medulloblastomas archived in the Neurological Surgery Tissue Bank at UCSF or the Neuropathology Laboratory at St. Jude Children’s Research Hospital. The St. Jude tumors were from a series reported previously 32 and have since been processed into tissue microarrays. We cut sections to 10-µm (UCSF) or 6-µm (St. Jude) widths and incubated them in 0.01 M citrate buffer (pH 6.0) at 95 °C for 30 min for antigen retrieval before immunostaining. We analyzed the samples to detect primary cilia blinded to the diagnosis or gene expression signature of the tumor. We used antibody to acetylated tubulin to detect primary cilia and antibody to γ-tubulin or pericentrin to detect the basal body. Statistical analyses. We used Student’s t test to compare BrdU+ cells in the embryonic EGL, the log-rank test to compare survival curves, the pairwise fixed relocation randomization test in Relative Expression Software Tool (REST) to compare qRT-PCR results and the Chi-square test to test the association of medulloblastoma subgroups and the presence or absence of primary cilia. 39. Zhuo, L. et al. hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo. Genesis 31, 85–94 (2001). 40. Pfaffl, M.W., Horgan, G.W. & Dempfle, L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acid Res. 30, e36 (2002).
doi:10.1038/nm.2020
