Sonic hedgehog-associated medulloblastoma arising ... - Springer Link

Acta Neuropathol (2012) 123:601–614 DOI 10.1007/s00401-012-0961-0

ORIGINAL PAPER

Sonic hedgehog-associated medulloblastoma arising from the cochlear nuclei of the brainstem Daniel Grammel • Monika Warmuth-Metz • Andre´ O. von Bueren • Marcel Kool • Torsten Pietsch • Hans A. Kretzschmar • David H. Rowitch Stefan Rutkowski • Stefan M. Pfister • Ulrich Schu¨ller

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Received: 7 November 2011 / Accepted: 13 February 2012 / Published online: 21 February 2012 Ó Springer-Verlag 2012

Abstract Medulloblastoma is a malignant brain tumor of childhood that comprises at least four molecularly distinct subgroups. We have previously described that cerebellar granule neuron precursors may give rise to the subgroup with a molecular fingerprint of Sonic hedgehog (Shh) signaling. Other recent data indicate that precursor cells within the dorsal brain stem may serve as cellular origins for Wnt-associated medulloblastomas. To see whether Shhassociated medulloblastomas are also able to develop in the dorsal brainstem, we analyzed two lines of transgenic mice with constitutive Shh signaling in hGFAP- and Math1positive brainstem precursor populations, respectively. Our results show that in both of these lines, medulloblastomas arise from granule neuron precursors of the cochlear nuclei, a derivative of the auditory lower rhombic lip. This region is distinct from derivatives of precerebellar lower rhombic

lip where medulloblastomas arise in mice with constitutive-active Wnt signaling. With respect to their histology and the expression of appropriate markers, Shh tumors from the murine cochlear nuclei perfectly resemble human Shh-associated medulloblastomas. Moreover, we find that in a series of 63 human desmoplastic medulloblastomas, 21 (33%) have a very close contact to the cochlear nuclei on MR imaging. In conclusion, we demonstrate that precursors of the murine rhombic lip, which either develop into cerebellar or into cochlear granule neurons, may give rise to Shh-associated medulloblastoma, and this has important implications for the cellular origin of human medulloblastomas. Keywords Medulloblastoma Sonic hedgehog Origin Brainstem Cochlear nuclei

Electronic supplementary material The online version of this article (doi:10.1007/s00401-012-0961-0) contains supplementary material, which is available to authorized users. D. Grammel H. A. Kretzschmar U. Schu¨ller (&) Center for Neuropathology, Ludwig-Maximilians-University, Feodor-Lynen-Strasse 23, 81377 Munich, Germany e-mail: [email protected] M. Warmuth-Metz Department of Neuroradiology, University of Wu¨rzburg, Wu¨rzburg, Germany A. O. von Bueren S. Rutkowski Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany M. Kool S. M. Pfister Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany

T. Pietsch Institute of Neuropathology, University of Bonn, Bonn, Germany D. H. Rowitch Eli and Edythe Broad Institute of Regeneration Medicine and Stem Cell Research and Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA, USA D. H. Rowitch Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA S. M. Pfister Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Heidelberg, Germany

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Introduction The knowledge on the cellular origin of a tumor is an increasing focus of oncology research. Such investigation promises a better understanding of the cell types within an organ that might be susceptible to oncogenic transformation as well information that could be used to develop novel targeted therapies. Such knowledge further appears useful in order to shed new light on the question whether multi-potent stem cells, lineage-committed precursor cells or fully differentiated cells may give rise to a tumor and how such tumor-initiating cells may be related to cancer stem cells with tumor-propagating properties. Finally, the identification of a tumor’s cellular origin will allow researchers to generate accurate mouse models that are urgently needed for preclinical studies of novel drugs (for a review, see [9]). Medulloblastoma (MB) is an embryonal neoplasm representing the most common malignant brain tumor in childhood. It has a strong tendency to metastasize along the cerebrospinal axis. Survival rates have significantly increased within the last 20 years, but many patients still die from this disease [19]. MB comprises four core subgroups that are defined by distinct molecular fingerprints from global gene expression arrays and that significantly differ with respect to genetics, histology and clinical outcome [23, 27, 33]. While two molecular subgroups are not clearly associated with deregulated signaling cascades, a third group is characterized by a constitutive activation of the Sonic hedgehog (Shh) pathway and a fourth group is known for its constitutive activation of Wnt/b-catenin signaling. The cellular origin of MB has attracted researchers’ interest for decades, but only recently it has been suggested that distinct molecular subtypes may have distinct cellular origins. As a first attempt to pin down the cellular origin of different medulloblastoma subtypes, we and others have used conditional mouse models to introduce mutations that constitutively activated the Shh or the Wnt signaling pathway in a spatially and timely controlled manner. Mutations were chosen accordingly to what had been described for human MB samples [12, 24] and, as a first candidate, Math1? cerebellar granule neuron precursors had been targeted by such mutations. In this sense, we have previously demonstrated that Math1? cerebellar granule neuron precursors may give rise to Shh-associated MBs, as revealed in Math1-cre::SmoM2-YFPFl/? and in Math1-cre::ptch1Fl/Fl animals [30, 39]. Using the same promoter, but mutations that would constitutively activate the Wnt signaling pathway (Math1-cre::ApcFlFl and Math1-cre::Ctnnb1(ex3)Fl/? animals) we found that oncogenic Wnt signaling in Math1? granule neuron precursors of the cerebellum did not lead to the formation of MB, but significantly inhibited their proliferation [15]. In fact, the

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cellular origin of the Wnt MB subgroup has now been suggested to be located within the dorsal aspect of the brainstem that is populated by precursor cells of the precerebellar rhombic lip normally giving rise to the pontine grey nuclei [8]. This, in general, raises the question to what extent cell populations outside of the cerebellum (e.g. precursors derived from the lower rhombic lip) may contribute to the formation of MB subtypes and we asked whether this could also be true for Shh-associated MBs. We show here that aberrant activation of Shh through a mutated Smoothened allele (SmoM2-YFP) in granule neuron precursors of the cochlear nuclei of the dorsal brainstem resulted in the formation of MB. Such tumors revealed a morphology and marker expression very similar to human Shh-associated MB. Furthermore, we provide evidence that the occurrence of human Shh-associated MB may not be restricted to the cerebellum. In fact, our results demonstrate that a subgroup of human MBs with a Shh profile has a very close contact to the cochlear nuclei within the dorsal brainstem suggesting that Shh-associated MB may have diverse cellular origins.

Materials and methods Mouse lines and genotyping ROSA26lacZFl/Fl [32], SmoM2-YFPFl/Fl [18] and hGFAPcre mice [40] were obtained from the Jackson Laboratory (Bar Harbour, ME, USA). Generation of Math1-cre mice has previously been described [20, 31]. Genotyping was performed through PCR according to previously published protocols from tail genomic DNA. Patients and tumor imaging Sixty-three consecutive patients registered to the HIT 2000 study (ClinicalTrials.gov/NCT00303810) with a histopathological diagnosis of desmoplastic medulloblastoma and with available magnetic resonance images (MRIs) of sufficient quality were used to investigate tumor localization and clinical aspects. Age, tumor location, metastatic and resection status as well as survival data are summarized in Suppl. Table 1. All MRIs were centrally reviewed at the National Reference Center for Neuroradiology (M. W.-M., Department for Neuroradiology, University Hospital of Wu¨rzburg, Germany) with pretreatment MRIs. Histopathological diagnoses of all cases were centrally confirmed at the Brain Tumor Reference Center, University Hospital of Bonn, Germany (T.P.), and were classified according to the World Health Organization classification of brain tumors [16]. Imaging was reviewed for the following items: localization of the medulloblastoma in the


hemispheres, the vermis, cerebello-pontine angle and the fourth ventricle, contact to the brain stem and especially the area of the cochlear nuclei. We tried to differentiate between the main localization and a local extension of the tumor. As a consequence of the multicenter nature of the study, the sequences varied widely, but routinely contained axial and sagittal T1- and T2-weighted slices performed on 1.5 Tesla (T) and rarely 3T-machines.

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working solution for 3 h, washed in PBS and counterstained in Kernechtrot. To co-visualize X-gal conversion by b-galactosidase with fluorescent dyes, a pseudo-color image was generated from the chromogenic, bright-field original. All histological photomicrographs were taken digitally using an Olympus BX50 microscope in combination with the Color view Soft imaging system. Immunohistochemistry and in situ hybridization

RNA extraction and gene expression profiling RNA from human and murine medulloblastoma tissue was extracted using TRIzolÒ reagent and following the manufacturer’s recommendations. Mouse tissue was then hybridized on Affymetrix mouse Genome 430 2.0 Genechips, while RNA from human medulloblastomas was analyzed using Affymetrix HG U133 Plus 2.0 Genechips, according to manufacturer’s instructions. Expression data were normalized using the MAS5.0 algorithm of the GCOS program (Affymetrix). Quality of the arrays was ensured by inspection of the beta-actin and GAPDH 50 -30 ratios as well as the percentage of present calls generated by the MAS5.0 algorithm (Affymetrix). Bioinformatics and biostatistics For comparison of clinical and pathological parameters, our tumor cohort (n = 63) was grouped into tumors with contact (n = 21) and without contact (n = 42) to the cochlear nuclei. Parameters between groups were compared using the Pearson0 s v2 test or the Fisher0 s exact test. Survival of patients was analyzed using Kaplan–Meier survival curves and the log-rank test was applied to examine the significance of results. Microarray data were analyzed using the R2 microarray analysis and visualization platform (http://www.r2.amc.nl). Histology and X-gal stainings For hematoxylin/eosin stainings and all immunohistochemical procedures, brains were dissected, fixed overnight in 4% paraformaldehyde at 4°C, embedded in paraffin, and sectioned at 5 lm according to standard protocols. Whole mount X-gal stainings of mouse brains were performed on freshly dissected samples according to the following protocol: samples were washed twice in PBS for 20 min and incubated in X-gal working solution [2 mg/ml X-gal (Peqlab), 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 2 mM MgCl2, 0.02% NP40 and 0.1% sodium deoxycholate in PBS] at room temperature for 2 h. X-gal stained samples were fixed in 4% paraformaldehyde at 4°C over night. For the detection of b-galactosidase in cryosections, unfixed material was equilibrated in PBS, incubated in X-gal

For immunohistochemistry, paraffin sections were subjected to heat antigen retrieval at 100°C for 20 min in 10 mM sodium citrate buffer for all antibodies. Chromogenic staining was performed using the HRP/DAB staining system (DAKO) according to the manufacturer’s specifications. Primary antibodies were against Ki67 (Tec-3, DAKO), NeuN (MAB377, Chemicon), Pax6 (aa1-223, Developmental Studies Hybridoma Bank), GFP (sc-8334, Santa Cruz) and Zic1 (gift from R. Segal). For fluorescent images, sections were incubated with secondary antibodies coupled to fluorescent dyes (Sigma) and counterstained with DAPI. Fluorescent images were taken on an Olympus IX50 microscope or a Zeiss LSM 510 confocal laser scanning microscope in combination with the Color view Soft imaging system. In situ hybridization with digoxigenin-labeled cRNA probes was performed as previously described [37] with the following modifications: Prior to pre-hybridization, sections were incubated in proteinase K solution (10 lg/ml in PBS) for 10 min at room temperature to improve RNA availability. Hybridization temperatures varied between 55 and 65°C depending on the used cRNA probe. Following hybridization, sections were washed in 50% formamide (in 19 SSC; 3° below hybridization temperature), incubated with RNaseA (2 lg/ml) for 15 min at 37°C and subsequently washed in decreasing SSC concentrations (29 SSC to 0.29 SSC; 3 degrees below hybridization temperature) for 1 h. For final color development, sections were incubated in BM purple (Roche) for up to 5 days.

Results Major cell populations of the dorsal brainstem derive from hGFAP? or Math1? precursors Recent data suggest that Wnt-associated MBs originate from precursors in the dorsal brainstem [8] and thus mark cell populations in this area as a novel cellular origin for MB. Apart from the tumorigenic potential of constitutive Shh activation in granule neuron precursors of the cerebellum [30], we asked to what extent precursor populations in the dorsal brainstem might be involved in the formation

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of Shh-associated MBs and might be susceptible to oncogenic transformation by constitutive Shh activation. To test this, we first aimed to assess the potential of hGFAP-cre mice to drive Cre-mediated recombination in populations of the dorsal brainstem as the hGFAP promoter is known to be active in neural precursors giving rise to glia and neurons throughout the entire brain [40]. Indeed, hGFAP promoter sequences were able to drive Cre recombination throughout large parts of the hindbrain as shown by whole mount X-gal staining of adult hGFAP-cre::ROSA26lacZFl/ ? mice (Fig. 1a, b). A detailed analysis within the dorsal brainstem revealed Cre-mediated recombination in neurons of the medial and lateral vestibular nucleus, the cochlear nuclei, the parasolitary nucleus, the external cuneate nucleus and the spinal trigeminal nucleus (Fig. 1d–f). Nevertheless, Cre recombination occurred only in subsets of the mentioned nuclei and was not detectable in other dorsal brainstem compartments, such as the area postrema or the nucleus tractus solitarii. To potentially cover additional cell populations, we included a Math1-cre driver in our analyses that has been known to drive recombination in neurons of various nuclei in the dorsal brainstem [6, 14, 17, 36]. In adult Math1-cre::ROSA26lacZFl/? mice, Cre-mediated recombination was detected in neurons of the medial and lateral vestibular nucleus, the cochlear nuclei, the nucleus tractus solitarius, the external cuneate nucleus, the area postrema and the spinal trigeminal nucleus (Fig. 1g– l). Even though various subpopulations of distinct dorsal brainstem nuclei seem to derive from hGFAP? as well as from Math1? precursors (e.g. granule cells of the cochlear nuclei), we note that the pattern of Cre-mediated recombination within single brainstem nuclei can greatly differ in hGFAP-cre and Math1-cre mice with regard to the number and shape of recombined cells. Taken together, hGFAP and Math1 promoter sequences allow Cre-mediated recombination in a variety of cell populations within major compartments of the dorsal brainstem. Constitutive Shh activation in hGFAP? and in Math1? precursors results in the formation of medulloblastoma within the cochlear nuclei of the dorsal brainstem To conditionally activate Shh signaling in precursor cells that establish nuclei of the dorsal brainstem, we generated hGFAP-cre::SmoM2-YFPFl/? and Math1-cre::SmoM2YFPFl/? mice, thereby introducing an activated Smoothened allele (SmoM2-YFP) in these cell populations [18]. As expected from previous studies [11, 25, 30], all mice with activated Shh signaling in hGFAP? and Math1? precursors developed diffuse tumors from granule cell precursors of the cerebellum that were readily visible from P0 (postnatal day 0) to P21 as compared to control animals (Fig. 2a–r). More interestingly, however, we further detected cellular

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hyperplasia within the region of the cochlear nuclei (CN) of the P0 brainstem (Fig. 2b, c, e, f; see also area depicted by arrows in Fig. 4k). These cells seemed to proliferate continuously, occupying a large part of the CN at P5 in hGFAPcre::SmoM2-YFPFl/? (Fig. 2h, k) and in Math1-cre::SmoM2-YFPFl/? mice (Fig. 2i, l). Note that tumor formation in the dorsal brainstem was restricted to the cochlear nuclei. At P21, tumors from the CN had markedly increased in size and infiltrated medially located brainstem tissue (Fig. 2n, o, q, r). The overall morphology was characterized by small, blue and round tumor cells, with high cell density and high similarity to human MB (see insets in Fig. 2q, r). Of note, no significant difference could be seen with regard to histology and localization of CN tumors deriving from either hGFAP? or Math1? precursor cells. Although we had proven that hGFAP-cre and Math1-cre animals caused genetic recombination both in cerebellar granule neurons as well as in the CN (Fig. 1), it appeared possible that tumor cells within the CN of hGFAP-cre::SmoM2-YFPFl/? and of Math1-cre::SmoM2-YFPFl/? mice would stem from diffuse infiltration of the cerebellar mass instead of building up an independent neoplasm. To address this question, we compared the hindbrains of adult hGFAP-cre::SmoM2-YFPFl/? to the hindbrains of ptch1?/- mice. Ptch1?/- mice represent a well established mouse model for medulloblastoma that develops brain tumors based on a clonal expansion of single cerebellar granule neurons that sporadically loose the second ptch1 allele [10]. As shown in Fig. 3a and b, both mouse models harbor large cerebellar tumor masses. Tumor cells within the CN, however, are exclusively seen in hGFAP-cre::SmoM2-YFPFl/? mice (Fig. 3c). In fact, mature granule cells are hardly detectable in hGFAPcre::SmoM2-YFPFl/? CN (Fig. 3c), whereas anatomical structures of the CN are well preserved in ptch1?/- mice (Fig. 3d). In line with this, the expression of NeuN revealed a gross disorganization of the CN in hGFAP-cre::SmoM2YFPFl/? mice (Fig. 3e), while the cochlear cortex in ptch1?/- mice (Fig. 3f) displayed the expected laminar structure [2]. Finally, we detected high numbers of cells expressing Ki67 in the CN of hGFAP-cre::SmoM2-YFPFl/? mice (Fig. 3g), but no proliferating cells in the CN of ptch1?/- mice (Fig. 3h). We therefore conclude that, in addition to cerebellar MB, constitutive activation of Shh signaling leads to independent neoplastic transformation and the formation of MB in hGFAP?/Math1? precursors of the cochlear nuclei. To seek for the cellular origin of medulloblastoma that arise within the CN, we then carefully analyzed the CN of newborn mice by combining fate mapping experiments with detection of cell type-specific markers. As shown in Fig. 4a–d, X-gal staining in hGFAP-cre::ROSA26lacZFl/? mice co-localized with Pax6, which had previously been shown to mark the granule cell lineage [6]. These analyses


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Fig. 1 Major cellular compartments of the dorsal brainstem were fate mapped by hGFAP-cre or Math1-cre. Fate mapping analyses of the dorsal brainstem were performed using ROSA26lacZFl/? reporter alleles and analyzed at postnatal day 21. Whole mount X-gal staining revealed successful recombination induced by both promoters throughout the entire hindbrain (a, g). Pictures in b and h display a dorsal view onto the brainstem. H&E-stained sagittal sections are shown for orientation purposes (c, i). X-gal stainings were performed on horizontal sections indicating functional Cre expression through hGFAP-cre promoter sequences in neurons of the dorsal and ventral cochlear nucleus (CN), the vestibular nucleus (VN; lateral: LVN; medial: MVN), the external cuneate nucleus (ECN), the parasolitary

nucleus (PSN) and the spinal trigeminal nucleus (STN). Insets show co-staining of X-gal (red) and NeuN (green) in the indicated nuclei (d–f). Math1-cre-induced genetic recombination in neurons throughout the entire cochlear nucleus (CN), the vestibular nucleus (VN; lateral: LVN; medial: MVN), the external cuneate nucleus (ECN), the nucleus tractus solitarius (NTS), the spinal trigeminal nucleus (STN) and the area postrema (AP). Insets show co-staining of X-gal (red) and NeuN (green) in the indicated nuclei (j–l). The table is showing a summary of fate-mapped cellular compartments in the dorsal brainstem for hGFAP-cre and Math1-cre promoter sequences (m). Scale bars 2,000 lm (a, g), 1,000 lm (b, c, h, i), 500 lm (d–f, j–l), 5 lm (insets in d–f, j–l)

indicated that granule neurons within the cochlear nucleus have an hGFAP? ancestry. At this age, some single Pax6? granule cells proliferated as revealed by co-labeling with Ki67 (Fig. 4e–g), while most cells expressed NeuN, but no

Ki67 indicating their maturity (Fig. 4h–j). High power views of horizontal sections through the CN of hGFAPcre::SmoM2-YFPFl/? mice first of all demonstrated that the CN tumor did not have any contact to the cerebellar mass

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Fig. 2 Constitutive Sonic hedgehog signaling resulted in the formation of medulloblastoma from granule cells of the cerebellum and of the cochlear nuclei. At P0, the cerebellar external granule cell layer and the region of the cochlear nuclei showed hypercellularity both in hGFAPcre::SmoM2-YFPFl/? (b, e) and in Math1-cre::SmoM2-YFPFl/? mice (c, f) when compared with control conditions (a, d). At P5 (g–l), tumors were well distinguishable for both genotypes in both anatomic locations, and at

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P21 (m–r), tumors were space occupying and diffusely infiltrating. All images were taken from H&E-stained paraffin sections. a–c, g–i and m–o are sagittal, d–f, j–l and p–r are horizontal. Dotted lines in n, o, q and r delineate the interface between brainstem and cerebellar tumors. Hemisections are shown in p, q and r (lateral is to the right). CN cochlear nuclei, EGL external granular layer, bMB brainstem medulloblastoma, cMB cerebellar medulloblastoma. Scale bars 500 lm (a–r)


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Fig. 3 Medulloblastoma cells within the brainstem represent independent neoplasms rather than infiltration by cerebellar masses. H&E stainings of sagittal sections of hGFAP-cre::SmoM2-YFPFl/? (a) and ptch1?/- mice (b) revealed massive tumor formation within the cerebellum of both mouse models. The formation of MB within the CN, however, was exclusively seen in hGFAP-cre::SmoM2-YFPFl/? mice (c), while the overall organization of the CN in ptch1?/- mice (d) remained unaffected. Arrows depict Golgi cells intermingled with tumor cells in c and in the FCL of the CN in d. Dashed line encircles the FCL. Expression of NeuN indicated a gross disorganization of the

CN of hGFAP-cre::SmoM2-YFPFl/? mice (e), whereas the laminar structure of the CN in ptch1?/- mice was well preserved (f). Tumor cells in the CN of hGFAP-cre::SmoM2-YFPFl/? mice displayed high proliferative activity as revealed by the expression of Ki67 (g), but we did not detect any proliferating cells within the CN of ptch1?/- mice (h). CN cochlear nuclei, FCL fusiform cell layer, GCD granule cell domain, bMB brainstem medulloblastoma, cMB cerebellar medulloblastoma, ML molecular layer. Scale bars 1,000 lm (a, b), 100 lm (c–h), 10 lm (insets in c, d)

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and therefore confirmed its independent origin (Fig. 4k). Second, activated Shh signaling in Pax6? cells of the microneuronal shell of the CN was documented by visualizing the mutant SmoM2-YFP protein (Fig. 4l–n). Finally, early tumor formation in hGFAP-cre::SmoM2YFPFl/? mice was characterized by highly increased proliferation of Pax6? cells and an accompanied expansion of the Pax6? cell population within the microneuronal shell of the CN (Fig. 4o–q). In contrast, the relative number of mature NeuN? and Ki67- neurons that are detectable in deeper parts of the CN dramatically decreased (Fig. 4r–t). Taken together, we conclude that Shh-induced MB formation in the brainstem has its origin in Pax6? granule cell precursors of the cochlear nuclei. The formation of Shh-induced MB from granule neurons in the cerebellum as well as the cochlear nuclei raises the question to what extent these two tumor entities differ from each other in terms of their gene expression profile. In a first step, we assessed gene expression for both tumor mouse models in cerebellar and cochlear tumor tissue. Tumors showed similar expression of markers for proliferation (Ki67, Fig. 5a, g, m, s), neuronal lineage (NeuN, Fig. 5b, h, n, t; Pax6, Fig. 5d, j, p, v and Zic1, Fig. 5e, k, q, w) and Shh activity (Nmyc, Fig. 5c, i, o, u) regardless of their cellular origin. Furthermore, all tumor tissues showed similar expression of Math1 mRNA, indicating a tumor origin in the rhombic lip (Fig. 5f, l, r, x). Although the expression of these markers did not reveal remarkable differences in these tumor entities, global gene expression profiles of cerebellar and cochlear tumor tissues for both tumor mouse models differed significantly. Using microarray technology, we found that 205 genes were significantly differentially expressed between the cerebellar and cochlear tumor tissues, regardless of the tumor mouse model (p \ 0.01). The top 50 differentially regulated genes are shown in Fig. 5y, and a full list of all significantly regulated genes is provided as Suppl. Table 2. These data again argue for different cells of origin for Shh-associated cerebellar and cochlear medulloblastoma in the mouse. The cochlear nuclei as a potential origin of human Shhassociated medulloblastoma To determine the significance that our findings might have for human MB, we assessed the localization of a large cohort of human desmoplastic MBs in MR images with respect to their localization within the posterior fossa and their proximity to the cochlear nuclei of the dorsal brainstem. We aimed to analyze the localization of desmoplastic MBs because of their clear over-representation in human Shh-associated MBs [4, 13, 23, 28, 34]. From all analyzed desmoplastic MBs (n = 63), 25 (40%) grew in the cerebellar hemispheres, 30 (48%) in the cerebellar vermis and 8

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Acta Neuropathol (2012) 123:601–614 Fig. 4 Sonic hedgehog-induced medulloblastoma arises from granule c cell precursors of the cochlear nuclei. H&E staining of the cochlear nuclei at P0 (magnification of Fig. 2d) displays granule neuron precursors that stem from derivatives of the lower rhombic lip and ensheath the cochlear nuclear complex (a) and such precursors were fate mapped by hGFAP-cre as revealed by X-gal stainings of hGFAPcre::ROSA26lacZFl/? brainstems (b, d), expressed Pax6 (c, d, f, g) and proliferated as shown by the expression of Ki67 (e, g). NeuNpositive, mature neurons within the CN did not proliferate (h–j). At the same age, hGFAP-cre::SmoM2-YFPFl/? mice showed increased cellularity in the microneuronal shell of the cochlear nuclei composed of granule cells (k), and such medulloblastoma cells showed activated Shh signaling as shown by YFP expression and expressed Pax6 (l–n). Indeed, the vast majority of proliferating cells expressed Pax6 (o–q), but no NeuN (r–t). DCN dorsal cochlear nucleus, EGL external granule cell layer, GCD granule cell domain, MB medulloblastoma, PC plexus choroideus, VCN ventral cochlear nucleus. Scale bars 250 lm (a, h), 100 lm (b, e, i, l; also valid for adjacent pictures), 5 lm (insets in d, g, k)

tumors (12%) involved midline and hemispheric structures (Fig. 6a; Suppl. Table 1). Patients with exclusively hemispheric tumors had a median age of 12.6 years as opposed to patients with midline/hemispheric tumors with a median age of 3.9 years and patients with tumors in the midline, who had a median age of 6.7 years. Within the entire tumor cohort (n = 63), 42 patients (67%) displayed a MB without contact to the CN, while 21 (33%) had contact to the CN as revealed by MR imaging (Fig. 6b; Suppl. Fig. 1). From these 21 tumors with contact to the CN, 19 tumors displayed a main localization in the midline or involved both midline and hemispheric structures. Only two tumors with contact to the CN were mainly localized in the cerebellar hemispheres. The median age of MB patients with contact to the CN was 6.9 years as opposed to a median age of 11.1 years for MB patients without contact to the CN. Analysis of the overall age distribution showed a highly significant difference between these two tumor groups (p B 0.0001), with a peak incidence of CN-associated MBs in children as compared to MBs without CN contact with a peak incidence in adolescents (Suppl. Fig. 1a). Further analyses of clinicopathological features of these two tumor groups like survival, metastatic status and residual disease did not show any statistically significant differences (Suppl. Fig. 1b–d). Figures 6c, d illustrate sagittal (Fig. 6c) and axial (Fig. 6d) MR images from a representative example for a midline tumor with close contact to the CN. Histological analysis of tumor tissue of this patient confirmed typical desmoplasia with pale islands in H&E staining (Fig. 6e) and internodular reticulin-positive fibers in Gomori silver staining (Fig. 6f). Furthermore, microarray data that was available for this case confirmed that this tumor was driven by constitutive Shh signaling. In particular, the 25 genes that had previously been identified as being most prominently expressed by Shh-associated MBs [13] were significantly upregulated as compared to other MB subgroups (Fig. 6g). Marker genes of the WNT group or of MB groups 3 or 4 were not


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Fig. 5 Shh-associated medulloblastomas of different origin similarly expressed markers of proliferation, neuronal lineage and Shh activity, but showed distinct global gene expression profiles. All tumors showed a proliferation rate of about 50% as revealed by immunostainings using antibodies against Ki67 (a, g, m, s). Neuronal lineage was illustrated by the expression of NeuN (b, h, n, t), Pax6 (d, j, p,

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Math1

v) and Zic1 (e, k, q, w). Expression of Nmyc indicated activity of the Shh signaling pathway (c, i, o, u) and expression of Math1 pointed to a tumor origin within the rhombic lip (f, l, r, x). Global gene expression profiling revealed distinct expression patterns of cochlear and cerebellar tumors. The 50 most differentially regulated genes are shown in a heatmap (y). Scale bar 50 lm (in a, for a–x)


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a

b

Contact of desmopl. MBs to the cochlear nucleus (n=63)

Distribution of desmopl. MBs (n=63)

Hemispheric tumors (n=25, median age= 12.6 years)

40%

48%

Midline tumors (n=30, median age =6.7 years)

33% 67%

12% Contact to cochlear nuclei (n=21, median age=6.9 years)

Tumors in midline and hemisphere (n=8, median age=3.9 years)

No contact to cochlear nuclei (n=42, median age=11.1 years)

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Fig. 6 Localization of human desmoplastic medulloblastomas. Gross neuroradiology reveals that 40% of the tumors grew in the cerebellar hemispheres, 48% were located in the midline and 12% involved both midline structures and hemispheres (a). Although 67% (n = 42) of the desmoplastic tumors had no contact to the cochlear nuclei, 33% (n = 21) displayed a close contact to the CN (b). As a representative

example, the tumor illustrated on sagittal (c) and axial (d) MR images has developed in a 2-year-old boy and appeared to erupt from the CN (arrows). H&E-stained sections displayed typical histology with intratumoral nodules (e), and Gomori silver staining confirmed internodular desmoplasia (f). Global gene expression profile from this tumor displayed constitutive Sonic hedgehog signaling (g)

significantly expressed. These findings indicate that a fraction of human Shh-associated MB has close contact to the CN and raise the question whether granule cells of the CN may also serve as a cellular origin for Shh-associated medulloblastoma.

brainstem cell populations, thereby providing new insights into the origins of murine Shh-associated MBs. We demonstrate here that hGFAP?/Math1? progenitors from the cochlear granule cell lineage were susceptible to oncogenic transformation by aberrant Shh signaling. Furthermore, we show that a subgroup of human desmoplastic MBs displayed close contact to the CN in the dorsal brainstem. The rhombic lip is a germinal epithelium at the dorsal edge of the hindbrain, the derivatives of which form parts of the cerebellum as well as of the brainstem [7, 36, 38]. While the upper rhombic lip (URL) generates different cell

Discussion In this study, we used hGFAP-cre and Math1-cre drivers to constitutively activate Shh signaling in various dorsal

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types of the cerebellum, the lower rhombic lip (LRL), divided into the auditory and precerebellar compartment, produces cells that form parts of the cochlear nucleus complex and the precerebellar system, respectively [5, 14, 17, 26]. So far, two different progenitor cell populations within the rhombic lip have been identified as possible origins for MB. Cerebellar granule neuron precursors that derive from the upper rhombic lip (rhombomere 1) have been demonstrated to give rise to Shh-associated MBs [30] and pontine grey neuronal progenitors emerging from rhombomeres 6-8 have been suggested as a source for Wntassociated MBs [8] (Fig. 7). These results, for the first time, experimentally proved that molecularly different types of MB may have distinct cellular origins. However, at least two questions were still left open at that point. First, MBs comprise four molecular subtypes (Shh, WNT, groups 3 and 4) [33], but cellular origins for tumors of subgroups 3 and 4 are completely unknown. Second, it was unclear, whether one molecular subgroup may have more than one cell of origin. While the origin of groups 3 and 4 MBs is still under intense investigation, we show here that, at least in mice, MBs of one molecular subtype, i.e. the Shh subtype, may have multiple cellular origins, namely cerebellar granule neurons and granule neurons of the CN (Fig. 7). Apart from our findings in transgenic mice, the analysis of human tumor samples demonstrated that tumors located in the midline of the posterior fossa are not necessarily MBs of the WNT subtype as suggested by Gibson and coworkers [6]. In fact, desmoplastic MBs (that usually belong to the Shh group) are not exclusively found in the cerebellar hemispheres. Our results reveal that 60% of desmoplastic MBs include midline structures and this

Fig. 7 Schematic drawing of the early rhombic lip with cells of origin for medulloblastoma. While Sonic hedgehog-associated medulloblastoma may arise from derivatives of the URL or the auditory RL, Wnt-associated medulloblastoma may have its origin in precursors deriving from the precerebellar RL. LRL lower rhombic lip, RL rhombic lip, URL upper rhombic lip

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confirms previous studies noting that 64% of desmoplastic MBs are located in the midline [29]. Interestingly, in the cohort described by Bu¨hren et al. [3], the fraction of desmoplastic MBs in the midline is much smaller, but, at the same time, median age of this cohort is[20 years of age as compared to 1.8 years of age in the study by Rutkowski [29] and 8.5 years of age in our present study. So, younger age is obviously associated with a higher tendency for desmoplastic medulloblastoma to occur in the midline, and this is also reflected in Fig. 6a. Although we were not able to detect any significant differences with respect to the overall survival of patients with Shh-associated MBs that did or did not involve the CN (Suppl. Fig. 1b), the preferred tumor incidence of MBs involving the CN in children might draw special attention of the neurosurgeon to the floor of the fourth ventricle during the removal of these tumors. We certainly acknowledge that desmoplastic histology and Shh gene expression profile do not coincidence in all cases and that the 21 cases of desmoplastic MB with a contact to the cochlear nucleus (Fig. 6b) could theoretically not carry a Shh profile. However, this seems highly unlikely given the fact that 20 out of 29 desmoplastic MB (69%) had a Shh profile in a recently published series of medulloblastoma [4]. In addition, gene expression analysis which was available for the representative case shown in Fig. 6 c and d indeed confirmed a Shh gene expression signature (Fig. 6e). The similarity of cerebellar and cochlear tumors in terms of histology and expression of particular markers (Fig. 5a–x) mirrors the analogy of cerebellar and cochlear granule neuron precursors [1, 21, 35], but it is interesting to notice that murine Shh MBs with different cellular origins are obviously distinct with respect to the overall gene expression profile (Fig. 5y). In this context, it is worth mentioning that a consensus study agreed on four main molecular subgroups for human MBs [33], but that there are already studies describing differences within one molecular subgroup. In particular, within the Shh subgroup, pediatric and adult cases were described to have molecularly distinct features [22], and it appears possible that different localizations or cellular origins within the Shh subgroup may also be paralleled by specific gene expression pattern. In summary, we provide several lines of evidence indicating that Shh-associated MBs may arise from multiple cellular origins. It remains unclear whether similar phenomenons may be observed in other MB subtypes, and larger studies on human MBs will be needed to further elucidate the clinical significance of distinct tumor localizations. Acknowledgments We are indebted to Severin Filser, Michael Schmidt, Veronika Kaltenbrunn and Silvia Occhionero for excellent

Acta Neuropathol (2012) 123:601–614 technical support as well as to Dr. Mehdi Shakarami and Julia Geyer for animal husbandry. We thank Dr. R. Segal (Harvard University, Boston, MA) for providing the anti-Zic1 antibody and ptch1?/brains. We also thank Peter van Sluis, Jan Koster, and Richard Volckmann (Academic Medical Center Amsterdam) for help with expression profiling. This work was supported by grants from the German Cancer Aid (Max-Eder-junior-research-program), the FritzThyssen-Stiftung and the Fo¨rderprogramm fu¨r Forschung und Lehre (Fo¨FoLe) at the Ludwig-Maximilians-University (all to US). M.K. is supported by Koningin Wilhelmina Fonds (grant no UvA-2011-4713) and KIKA. The HIT-trial-office at the University Medical Center Hamburg-Eppendorf is supported by the German Children’s Cancer Foundation (Deutsche Kinderkrebsstiftung) and by the Fo¨rdergemeinschaft Kinderkrebszentrum Hamburg e. V.

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