Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma.
Journal: 2008/August - Cancer Cell
ISSN: 1878-3686
Abstract:
Whether the brain tumor medulloblastoma originates from stem cells or restricted progenitor cells is unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted central nervous system (CNS) progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNPs) derive from hGFAP(+) and Olig2(+) rhombic lip progenitors. Hh activation in a spectrum of early- and late-stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors.
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Cancer Cell 14(2): 123-134

Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Hedgehog-induced medulloblastoma

+6 authors

Significance

Cancer stem cells have been proposed as therapeutic targets in human brain cancer. Although such populations have been reported in medulloblastoma, tumor progenitor origins remain unclear. Here we show that medulloblastoma (MB) is generated from cells related to several stages of cerebellar granule neuron precursor (CGNP) development, and that some tumor cells, demonstrable in murine models and human MB, retain primitive features analogous to precursors of the embryonic brain. These data highlight the importance of a developmental perspective in understanding cells involved in CNS tumor progression and the histopathological composition of brain cancers. They suggest that a potential therapeutic target is the process regulating acquisition of CGNP phenotype.

Introduction

It has been proposed that cancer can be viewed as aberrant organogenesis initiated by a multipotent or restricted progenitor cell that acquires infinite capacity for self-renewal through accumulated mutations (Reya et al., 2001). Principles of normal hematopoietic stem cell development (Barabe et al., 2007; Cobaleda et al., 2007) have provided insight into the mechanisms of self-renewal and multi-lineage differentiation in hematopoietic malignancies (Faber and Armstrong, 2007; Warner et al., 2004). However, for most organ systems parallels between normal stem cell development and solid tumor formation remain poorly understood.

The central nervous system (CNS), with its highly diversified classes of neuronal and glial progenitor cells, provides a suitable model to study such mechanisms. Several studies have documented the existence of cancer stem cells for glioma and the cerebellar tumor, medulloblastoma (MB) with properties reminiscent of normal neural precursors (Singh et al., 2004; Galli et al., 2004; Hemmati et al., 2003). Expression profiling has indicated correlations between specific stages of cerebellar development and MB (Lee et al., 2003; Kho et al., 2004). The Sonic hedgehog (Shh) pathway has critical functions in cerebellar development and mutations of pathway are etiologic in MB (Pietsch et al., 1997).

The cerebellum (CB) develops from several progenitor regions: the rostral rhombic lip (rRL) and cells of the ventricular zone (VZ) surrounding the IVth ventricle (Altmann and Bayer, 1997; Morales and Hatten, 2006; Kawauchi et al., 2006; Casper and McCarthy, 2006; Malatesta et al., 2003) (Figure 1A). The VZ of the cerebellar anlage generates Purkinje neurons (PN), interneurons (IN), and glia cells. Progenitors within the rRL express the radial glial markers, RC2 and Nestin, and give rise to all granule lineage cells (GC) of the EGL and IGL. We recently reported that cells which express cre recombinase under control of human regulatory sequences for Glial Fibrillary Acidic Protein (hGFAP), are most probably radial glia and generate most cerebellar cell types including CGNP (Spassky et al., 2008). Subsequently, CGNP migrate rostrally along the surface of the cerebellar anlagen to form the external granule layer (EGL) (Lee et al., 2005; Machold and Fishell, 2005; Wang et al., 2005) (Figure 1A).

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Identification of an Olig2 progenitor population in the rostral RL

(A) The cerebellar anlagen at E14. The VZ and RL serve as mitotic niches at this age. The EGL is formed by proliferating CGNP that have left the RL. (B) At P7, the developing CB is divided into 10 lobes with defined cortical layers. (C-E) Sagittal sections at E14 in hGFAP-cre mice showing co-localization of Nestin, cre and Pax6 proteins in progenitor cells of the VZ and rRL. (F) Olig2 expression is largely restricted to dorsal rRL. (G, H) Fate mapping experiments (hGFAP-cre × ROSA26-eYFPFl/Fl mice) reveals YFP in the EGL and a subset of RL cells that express Olig2 (arrows). (I) Olig2 RL precursors co-label with the CGNP marker Pax6; some single Pax6 precursors in the VZ were observed (arrows). (J) Summary of cells in the RL region at E14 showing relative positions of radial glia (yellow/green), Pax6(blue) , Olig2(red) and Pax6Olig2(purple) cells. BG, Bergmann glia; CP, choroid plexus; GNP, granule neuron precursors; ML, molecular layer; PL, Purkinje cell layer.

PN (Figure 1B) produce Shh, which binds the transmembrane protein repressor Patched (Ptc), relieving inhibition of Smoothened (Smo) activity, which is essential for CGNP proliferation in the EGL (Wechsler-Reya and Scott, 1999; Dahmane and Altaba, 1999; Lewis et al., 2004). CGNP of the EGL gradually show reduced proliferation levels, migrate internally and differentiate into glutamatergic neurons in the internal granule layer (IGL) (Figure 1B), a process that is largely completed by postnatal day 15 (P15) in mice (Borghesani et al., 2002), and by about a year of age in the human CB (Abraham et al., 2001). Transcriptional control of CGNP development involves functions of bHLH protein Math1 (Ben Arie et al., 1997; Machold and Fishell, 2005; Wang et al., 2005), and homeodomain protein Pax6 (Engelkamp et al., 1999; Yamasaki et al., 2001).

Inherited activating mutations of the Shh-Smo pathway in humans are etiologic in MB but not glioma (Hahn et al., 1999; Johnson et al., 1996). This cancer spectrum is faithfully modeled in the CNS of mice heterozygous for a mutation of the Hedgehog (Hh) pathway suppressor Ptc1 (Johnson et al., 1996) or expressing an activated allele of Smoothened (SmoM2) (Mao et al., 2006), supporting the proposal that CGNP are particularly susceptible to transformation by oncogenic Hh signaling (Shih and Holland, 2004). Nevertheless, the cellular origins of MB from unipotent precursors in the EGL remain controversial. The identification of multipotent MB stem cells (Hemmati et al., 2003; Singh et al., 2004) capable of multi-lineage differentiation, in particular, has challenged the hypothesis of a unipotent cell-of-origin. To investigate these issues, we defined distinct progenitor populations for CGNP within the rRL as well as in the EGL and systematically assessed their tumorigenic potential with respect to Hh signaling.

Results

Olig2 expression identifies RL progenitors for a subset of granule cell neurons

In order to determine possible parallels between progenitors during cerebellar development and tumorigenesis, we first characterized relevant embryonic hindbrain VZ and RL populations. Nuclei of cells that express cre proteins in hGFAP-cre mice are observed along the VZ and the ventral aspect of the rRL of the midline cerebellar anlage at E14.5 (Figure 1C-E). Cre proteins in these mice mark nestin cells that have been proposed to be radial glia of the rRL that give rise to CGNP (Spassky et al., 2008), which show robust Pax6 expression. We next investigated expression of the bHLH protein, Olig2, which marks multilineage progenitor populations of the forebrain and the spinal cord (Petryniak et al., 2007; Lu et al., 2002; Zhou and Anderson, 2002). Olig2 expression identified a distinct cell population predominantly restricted to the rRL and extending to the boundary of the EGL (Figure 1F). The cerebellar VZ contained rare Olig2 cells and expression of Olig2 was rapidly down regulated within the EGL.

To establish a possible lineage relationship between hGFAP-cre and Olig2 cells, we fate mapped hGFAP-cre-expressing precursors with a conditional ROSA26-eYPFFl/Fl reporter (Srinivas et al., 2001). As shown (Figure 1G, white line), hGFAP-cre-expressing precursors give rise to the majority of CGNP of the primitive EGL, and some Olig2 cells of the rRL (Figure 1H). Most Olig2 cells in the rRL co-expressed the CGNP marker, Pax6 (Figure 1I) while Olig2 expression was gradually lost and Pax6 retained as CGNP migrated into the EGL. Olig2Pax6 cells could be identified within the rRL and EGL until E18.5, but were never observed at P7 or later stages (data not shown; Figure 6E,F).

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Olig2- and Gfap-expressing cells in medulloblastoma are neoplastic and have features in common with immature granule lineage precursors

(A, C) In Math1-cre:SmoM2 animals, morphologic evidence hyperplasia is predominantly observed in the EGL, but not RL or VZ, at P0 and is more apparent by P7. (B, D) Greatly increased numbers of Olig2 cells were observed in hyperplastic regions of the EGL--but not RL--of P0 and P7 Math1-cre:SmoM2 mice. (E, F) Expression of cre proteins in Math1-cre mice co-localized with Olig2 at E18 but not P7. (G) Olig2 co-expression was detected in only ∼2% of pH3 cells in tumors. (H) In contrast, ∼98% of pH3 cells co-labeled with cre proteins. (I-K) Phenotype of acutely disassociated cells and (insets) cells cultured from MB for four weeks. While most of dissociated Math1-cre:SmoM2 tumor cells express the GC markers Pax6 or (inset) NeuN (I), some express Olig2 (J) and Gfap (K). SmoM2 is fused to YFP, which therefore marks tumor cells. (L) Cell counting reveals that more than 90% of cells expressing Gfap, Olig2 and Pax6 are tumor cells, as shown by co-expression of YFP. (M, N) Immunohistochemistry of tumor sections demonstrates similar results. (O) 81% of Olig2 and (P) 100% of Gfap cells in tumors expressed the CGNP marker Pax6.

To establish Olig2 progenitor contributions during CGNP development, we constructed a multi-functional mouse line by inserting tva, an avian-specific retroviral receptor, and an IRES-cre recombinase cassette into the endogenous Olig2 locus by homologous recombination (Figure 2A). When Olig2-tva-cre mice were crossed to ROSA26-eYFPFl/Fl conditional reporter mice, we found that 98% of YFP cells in the P7 cerebellar white matter (WM) expressed Olig2, consistent with oligodendroglial identity (Figure 2B, C, yellow arrows). At P21 most of these cells co-expressed myelin basic protein, and we observed small numbers of YFP PN and IN (Supp. Figure 1E, F). At P7, we detected a small subpopulation of YFP CGNP within the EGL, confined mainly to posterior cerebellar Lobes IX/X (Figure 2C-E, white arrows). At P21, we observed fate mapping to GC within the IGL (Figure 2F-I). Interestingly, only 3% of YFP cells expressed the GC marker Zic in the Lobe II IGL, but 58% of YFP cells were double positive in Lobe X (Figure 2G and I), indicating that Olig2 contributions to GC of the IGL are restricted mainly to the posterior lobes.

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A subset of cerebellar granule neurons derive from Olig2 progenitors

(A)Olig2-tva-cre allele and genotyping of the respective mutant mice using PCR to generate wild type 429bp and mutant (cre) 446bp products. (B-I) Fate mapping of Olig2-expressing cells with ROSA26-eYFPFl/Fl reporter. Co-localization of YFP and Olig2 proteins in sagittal sections of P7 cerebella reveals that YFP is expressed in oligodendrocytes within the cerebellar WM (yellow arrows in C) as well as in occasional cells within the EGL (white arrows). YFP-Pax6 labeling confirms the granule lineage character of YFP-expressing EGL cells (D, E, arrows). The IGL of P21 cerebella demonstrates lobe-specific differences in Olig2 fate mapping. (G) The IGL of Lobe II contains YFP cells that predominantly co-express Olig2 (arrow and cell shown, inset) but not the GC marker Zic. In contrast, the IGL of Lobe X (H) contains numerous YFP/Zic double-positive cells (arrow and inset). (I) Mean percentage (+/-SEM) of Olig2YFP and Zic1YFP double-positive cells in the IGL of lobe II and X. (J-M) In utero fate mapping of Olig2-expressing cells by RCAS-GFP virus injection into the rRL of E15.5 Olig2-tva-cre mice. Analysis of the CB at P7 reveals GFP to be expressed in Olig2 (K) WM cells, as well as Pax6 (L) and Zic (M) cells of the IGL in Lobe X.

We found Olig2 cells in the rRL from E14.5-18.5 (Figure 1 and data not shown). To test whether these cells in particular were a source of lobe IX-X CGNP, we injected the rRL of E15.5 Olig2-tva-cre animals with an avian retrovirus (RCAS-GFP), which can only transduce tva-expressing cells (Figure 2J). Fate mapping results analyzed at P7 indicated that infected rRL Olig2-tva cells generate Pax6 CGNP of the EGL and Zic GC within the IGL (Figure 2K-M, Suppl. Figure 1) of lobes IX and X. Taken together, these data indicate that Olig2 progenitors of the rRL make restricted contributions to the GC lineage, particularly in posterior lobes of the CB.

Smoothened activation within CNS hGFAP and Olig2 progenitors produces only medulloblastoma

To determine the significance of hGFAP- and Olig2-expressing progenitor populations for CNS tumorigenesis, we used hGFAP-cre and Olig2-tva-cre drivers, respectively, to conditionally express an activated Smo (SmoM2) allele fused with yellow fluorescent protein (YFP) sequences (Mao et al., 2006). All hGFAP-cre:SmoM2 mice developed diffuse tumors with the characteristic “small round blue cell” histology of MB (Figure 3A-D, Table 1). Olig2-tva-cre:SmoM2 mice developed tumors with similar histology, except that they were focal, localized to the posterior-lateral lobes and had significantly (p <0.01) later mortality (Figure 3E-H, Table 1), which was likely a result of their focal nature and the small numbers of CGNP targeted by Olig2-tva-cre.

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Development of medulloblastoma but not glioma from hGFAP and Olig2 precursors

Targeting hGFAP and Olig2 precursor cells with SmoM2 results in MB (A-H). While neoplastic transformation of hGFAP cells affects the entire cerebellar cortex (A-D), MBs from Olig2-expressing precursors are restricted to posterior cerebellar regions (E-H). Although hGFAP-cre and Olig2-cre are broadly expressed in all parts of the CNS, neoplastic lesions due to SmoM2 are restricted to the CB. Forebrain sections including the subventricular zone (SVZ) in adult animals appear normal (I-L). Transplantation of Olig2-tva-cre:SmoM2 tumor cells into SCID mice gives rise tumors both in hindbrain (M,N) and forebrain (O,P) regions. A, E and I-P are frontal, B-D and F-H are sagittal sections stained with H&amp;E. C and D are high power magnifications from B, so are G and H from F. Asterisks mark lateral ventricles in I, K and O and the IV ventricle in M. BS, brainstem.

Although hGFAP and Olig2 progenitor populations give rise, collectively, to CGNP as well as IN, astrocytes (AS) and oligodendrocytes (OL), we only observed cerebellar tumors with histological features of MB. Additionally, activated Hh signaling in cerebellar PN driven by Shh-creERT2 never resulted in MB (Table 1). These data demonstrate that hGFAP and Olig2 multipotent progenitor populations can produce MB.

Hh signaling can have mitogenic effects in spinal cord and forebrain (Mao et al., 2006; Rowitch et al., 1999; Fuccillo et al., 2006), and roles in forebrain glioma have been reported (Clement et al., 2007; Ehtesham et al., 2007). However, no evidence of gliomas or other primitive neuroectodermal tumors (PNET) in the forebrain was observed despite forced expression of SmoM2 in the subventricular zone, in NG2 progenitors, and in oligodendrocyte precursor cells (OPC) of the forebrain (Table 1, Figure 3I-L). Together, these data indicate a hindbrain- as well as a CGNP/lineage-restricted oncogenic response to Hh signaling, and raised the question of whether this was determined by the particular environmental niche of the EGL. To assess this, we transplanted freshly dissociated cells from Olig2-tva-cre:SmoM2 tumors into the forebrains and cerebella of SCID mice. Because tumors formed equally well in both locations (Figure 3M-P, Table 1), suggesting tumor propagation is governed primarily by cell-intrinsic factors rather than environment.

Unipotent CGNP are competent to produce medulloblastoma

In myeloid oncogenesis, multipotent cells rather than more restricted progenitor cells are thought to be the origin of leukemia stem cells (Faber and Armstrong, 2007). To test whether a restricted neuronal precursor can also give rise to MB, we activated SmoM2 in committed CGNP. Math1-cre drives expression within CGNP of the rRL and the entire EGL until P7-10, but not in PN, AS or IN (Machold and Fishell, 2005; Wang et al., 2005). In contrast, expression of the transcription factor Tlx3 (Hox11L2) is only seen in the EGL of Lobes VI-IX, but never in other lobes or the rRL (Suppl. Figure 2). Transgenic Math1-cre mice were generated using regulatory sequences 4 kb upstream of Math1 (Matei et al., 2005; Schüller et al., 2007) and Tlx3-cre mice were generated by gene targeting of Tlx3 (Xu et al., 2008). Fate mapping crosses involving a conditional ROSA26-eYFPFl/Fl reporter confirmed that Math1 and Tlx3 precursors contributed solely to the GC lineage (Figure 4A-H and data not shown).

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Late stage unipotent CGNP are competent to produce medulloblastoma

(A-H) Cre expression driven by Math1 or Tlx3 promoter sequences is normally restricted to granule lineage cell as shown by crosses with Rosa26-eYFP conditional reporter mice. (C, D, G) GC fate-mapped by Tlx3-cre are restricted to Lobes VI-IX. (F, H) In contrast, Calbindin-expressing PN do not derive from Math1 or Tlx3 precursors. (I-L) Both Math1 and Tlx3 granule neurons give rise to MB after activation of SmoM2, and Tlx3-cre driven tumors obeyed posterior restriction consistent with fate mapping (compare arrows in C, K).

When crossed with SmoM2Fl/Fl mice, all Math1-cre animals developed MBs that could be transplanted into the cerebella of SCID mice (Figure 4I, J, Table 1, also see (Yang et al., 2008)). Tlx3-cre:SmoM2 animals developed tumors in a posterior distribution in accord with the Tlx3 expression domain (Figure 4K, L). These data show that unipotent CGNP are capable of tumor formation when targeted with oncogenic SmoM2. The Shh transcriptional target, Gli1, is expressed in proliferating CGNP (Wechsler-Reya and Scott, 1999) and we could induce MB formation in Gli1-creERT2:SmoM2 mice until P14, but not at later postnatal stages.

Medulloblastomas exhibit a common phenotypic endpoint despite diverse cellular origins

To determine whether significant cellular (i.e. glial and neuronal diversity) and gene expression differences might exist in tumors derived from multipotent (e.g. hGFAP-cre) versus unipotent (e.g. Math1-cre) progenitor populations we performed Affymetrix gene expression profiling analyses, previously demonstrated to aid in distinguishing MB from other histologically similar primitive neuroectodermal tumors (Pomeroy et al., 2002). Comparison of expression profiles of the different murine tumors and Ptc medulloblastoma using principal component (PC) analysis confirmed that all tumors had a gene expression signature of medulloblastomas and were highly related to each other (Figure 5A). Along the PC1 coordinate — the direction of greatest transcriptomic sample variance in cerebellar development —centroids of the MB tumor profiles were significantly different from matched normal samples (p < 10). However, PC1 coordinate centroids were not significantly different for tumors derived from early (i.e., hGFAP- or Olig2-cre) versus late/unipotent (i.e., Math1- or Tlx3-cre) GC lineage progenitors (Suppl. Figure 3). In fact, only a very small number of genes (∼150 of 54,675 probes tested) were found to be uniquely and differentially expressed in each tumor sub-group. In line with this, extensive histological (Figures 3C, 3G, 4J, 4L) and immunohistochemical analysis (Figure 5B) failed to reveal any clear differences in the cellular composition of the tumors. Furthermore, hierarchical cluster analysis based on the 150 genes differentially expressed amongst the tumors did not show strict clustering of tumors based on cell of origin (Suppl. Figure 4). Together, these findings indicate that tumors derived from early (i.e., hGFAP-cre or Olig2-cre) or late/unipotent (i.e., Math1-cre or Tlx3-cre) GC lineage progenitors arrive at a common phenotypic endpoint.

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Medulloblastoma generated from diverse progenitor cell origins leads to a convergent phenotypic endpoint

(A) Principal component analysis of global gene expression in conditional MB models [Math1(green)-, Tlx3(magenta)-, hGFAP(red)-, Olig2(blue)-cre:SmoM2 and Ptc (turquoise)] and samples from normal littermate control CB (black circles) are mapped onto normal cerebellar developmental space as defined by a 7000 member, rank-normalized gene set (Kho et al., 2004), from developing CB at stages P1, 3, 5, 7, 10, 15, 21, 30, 50 and 60 (orange numbers). Tumors were related to early rather than late developmental stages of the CB as we have reported for human MB (Kho et al., 2004). (B) Immunohistochemical analysis confirms all murine tumors exhibit a similar immunohistochemical staining pattern to each other as well as human MB with respect to standard neuronal and glial neuropathological markers. Results are representative of at least two tumor samples.

Cells expressing Gfap and Olig2 in Hh-activated medulloblastomas are neoplastic

All mouse tumors analyzed showed histological features in common with some human MBs including expression of CGNP markers (Math1, Zic, Pax6), Gfap, a marker usually found to be expressed in astrocytes, and Olig2, which is required for OL development (Lu et al., 2002) (Figure 5C). Clinical symptoms of tumor burden in Math1-cre:SmoM2 tumors developed at an average age of 41 days (Table 1), however we found evidence of hyperplasia in the EGL at P0 and more clearly at P7 (Figure 6A, C). Strikingly, high expression of Olig2 was also found in these regions (Fig 6B, D). At P7, when the EGL is clearly discerned, excessive numbers of Olig2 cells were observed in the EGL but not RL of Math1-cre:SmoM2 mice (Figure 6D). Indeed, although Olig2 cells that co-expressed Pax6 (Figure 1I) or a Math1 proxy marker (Figure 6E) could be observed at antenatal stages until E18, such co-labeled cells were not detectable at P7 (Figure 6F) or later stages. Thus, persistent Olig2 expression in the EGL of Math1-cre:SmoM2 mice represents an early molecular marker of a pre-neoplastic state.

We found that only about 2% of mitotically active phosphoH3 (pH3) cells were Olig2 in fully developed Math1-cre:SmoM2 tumors (Figure 6G). Indeed, the vast majority of proliferating cells were Cre (Figure 6H), a proxy for Math1 expression, indicating that most cells contributing to MB growth are CGNP-like. In human tumors it is controversial whether cells expressing GFAP (or OLIG2) comprise tumor, or alternatively, represent intermingled cells from the normal brain. This is a critical point given that glial differentiation within MB would provide further evidence for a multipotent stem cell-like progenitor. To establish the nature of cells comprising MB, we dissociated Math1-cre:SmoM2 tumors and found that the SmoM2-YFP fusion protein, a marker of cells bearing the tumorigenic mutation, co-localized in cells expressing either Pax6, NeuN, Gfap or Olig2. Quantification revealed that YFP proteins co-localized with most if not all Pax6, Olig2 and Gfap (100%, 90%, 96%, respectively) cells in acutely dissociated tumors (Figure 6I-L). In addition, we identified Olig2 and Gfap cells that co-expressed the YFP reporter in vivo (Figure 6M, N). Thus, Olig2- and Gfap cells are tumor cells rather than infiltrated/normal glial cells.

The Gfap and Olig2 cells might represent simple astroglial and oligodendroglial tumor components. However, examination of Gfap and Olig2 cells of the cerebellar anlagen (Figure 1) suggested that such tumor cells could, alternatively, retain features of primitive CGNP. In support of the latter possibility, we found that 81% of Olig2 cells expressed Pax6 and that 100% of Gfap cells expressed Pax6 in acutely disassociated Math1-cre:SmoM2 tumors in vitro (Figure 6O, P). These findings indicate that cells with an immature/progenitor phenotype are present in Hh-activated MB.

Human medulloblastoma contain rare OLIG2 cells with progenitor features

In order to determine the significance our findings might have for human MB, we examined OLIG2 cell populations within a cohort of human MBs. Although our original findings in mice were all in the setting of Shh/Smo pathway activation, we found that OLIG2 cells were present in both classic (40%) as well as desmoplastic (Shh-related) (77%) subtypes of human MBs (Fig 7A-D; Suppl. Table 1), and comprised less than 5% of cells in all tumors. Fluorescent in situ hybridization (FISH) analysis showed that OLIG2 cells in human MB contained copy number gain at the C-MYC locus, confirming them as tumor cells and arguing against the possibility that they might represent intermingled normal cells (Figure 7E, F). As further support for OLIG2 cells being tumor cells we identified co-expression of OLIG2 and the terminal neuronal differentiation marker, NEUN, in rare cells within human tumors (Figure 7G), and we found rare OLIG2 cells that expressed the proliferation marker, Ki67 (Figure 7H). Together, these data suggest the possibility that some OLIG2 cells are tumor cells with immature features and may represent a type of MB progenitor cell.

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OLIG2 is expressed in human medulloblastoma

(A-D) OLIG2 expression in human classic and desmoplastic MB and percentage of tumors of each class that contain OLIG cells (also see Suppl. Tab 1). (E, F) Combined FISH and immunohistochemical analysis reveals the presence of tumor specific C-MYC genomic aberrations (Herms et al., 2000) within OLIG2 cells (yellow nuclei) of human MB (n=2). C-MYC aberrations were identical in OLIG2 and OLIG2 tumor cells. Note combined copy number gain (red signals, 5′ C-MYC) and structural aberration (loss of green signals, 3′ C-MYC), as detected using break apart probe set. Normal MYC loci show merged colors as (focal yellow signals). (G, H) OLIG2 co-localizes with NEUN, and the proliferation marker Ki67. (I) Scheme for experiments using RCAS-GFP infection of Olig2-tva-cre:SmoM2 tumor cells. (J-M) Double-fluorescence images show expression of GFP that co-localizes with immunoreactivity using antibodies against Olig2 and GC lineage markers. Note that while Olig2-tva-cre:SmoM2 express YFP the signal is very weak/undetectable compared with GFP encoded by RCAS virus, as shown (J-M). (N) Summary of findings at Day 6 in GFP-labeled cells. (O-Q) Analysis of acutely disassociated Olig2-tva-cre:SmoM2 tumor (Day 0). Of Olig2+ cells, 66% co-labeled with Pax6 and 12% with NeuN, such cells represented 4% and 1% of total cells in the tumor, respectively.

To mechanistically assess the lineage differentiation potential of Olig2 cells in the context of MB, we acutely disassociated tumors from Olig2-tva-cre:SmoM2 mice and infected these cultures with RCAS-GFP viruses, to perform fate mapping of tumor stem/progenitor cells (Figure 7I). In our experimental paradigm, cells must be Olig2-tva for infection with RCAS-GFP to occur. After six days in culture, we found that cells derived from Olig2 progenitors expressed Pax6, Zic or NeuN (Figure 7K-N). Significantly, of the 561 GFP cells counted, only 68 (12%) expressed Olig2 on Day 6 (Figure 7J, N), indicating differentiation along GC lineage axis with down-regulation of Olig2. Upon acute harvest and disassociation of tumors (Day 0), we found that only 8% expressed Olig2. Of these, 66% co-expressed Pax6 and 12% expressed NeuN (Figure 7O-Q). Contrasting this, on Day 6 in the tva fate map experiment, 52% of Olig2-derived progeny expressed NeuN. These data are consistent with the proposal that Olig2Pax6 cells lose Olig2 expression and progress to express the more mature markers such as Zic and NeuN during the 6 days in culture. We conclude that Olig2 cells in Olig2-tva-cre:SmoM2 tumors have the potential to produce GC-like progeny therefore supporting the possibility that they represent a subset of MB progenitor cells.

Olig2 expression identifies RL progenitors for a subset of granule cell neurons

In order to determine possible parallels between progenitors during cerebellar development and tumorigenesis, we first characterized relevant embryonic hindbrain VZ and RL populations. Nuclei of cells that express cre proteins in hGFAP-cre mice are observed along the VZ and the ventral aspect of the rRL of the midline cerebellar anlage at E14.5 (Figure 1C-E). Cre proteins in these mice mark nestin cells that have been proposed to be radial glia of the rRL that give rise to CGNP (Spassky et al., 2008), which show robust Pax6 expression. We next investigated expression of the bHLH protein, Olig2, which marks multilineage progenitor populations of the forebrain and the spinal cord (Petryniak et al., 2007; Lu et al., 2002; Zhou and Anderson, 2002). Olig2 expression identified a distinct cell population predominantly restricted to the rRL and extending to the boundary of the EGL (Figure 1F). The cerebellar VZ contained rare Olig2 cells and expression of Olig2 was rapidly down regulated within the EGL.

To establish a possible lineage relationship between hGFAP-cre and Olig2 cells, we fate mapped hGFAP-cre-expressing precursors with a conditional ROSA26-eYPFFl/Fl reporter (Srinivas et al., 2001). As shown (Figure 1G, white line), hGFAP-cre-expressing precursors give rise to the majority of CGNP of the primitive EGL, and some Olig2 cells of the rRL (Figure 1H). Most Olig2 cells in the rRL co-expressed the CGNP marker, Pax6 (Figure 1I) while Olig2 expression was gradually lost and Pax6 retained as CGNP migrated into the EGL. Olig2Pax6 cells could be identified within the rRL and EGL until E18.5, but were never observed at P7 or later stages (data not shown; Figure 6E,F).

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Olig2- and Gfap-expressing cells in medulloblastoma are neoplastic and have features in common with immature granule lineage precursors

(A, C) In Math1-cre:SmoM2 animals, morphologic evidence hyperplasia is predominantly observed in the EGL, but not RL or VZ, at P0 and is more apparent by P7. (B, D) Greatly increased numbers of Olig2 cells were observed in hyperplastic regions of the EGL--but not RL--of P0 and P7 Math1-cre:SmoM2 mice. (E, F) Expression of cre proteins in Math1-cre mice co-localized with Olig2 at E18 but not P7. (G) Olig2 co-expression was detected in only ∼2% of pH3 cells in tumors. (H) In contrast, ∼98% of pH3 cells co-labeled with cre proteins. (I-K) Phenotype of acutely disassociated cells and (insets) cells cultured from MB for four weeks. While most of dissociated Math1-cre:SmoM2 tumor cells express the GC markers Pax6 or (inset) NeuN (I), some express Olig2 (J) and Gfap (K). SmoM2 is fused to YFP, which therefore marks tumor cells. (L) Cell counting reveals that more than 90% of cells expressing Gfap, Olig2 and Pax6 are tumor cells, as shown by co-expression of YFP. (M, N) Immunohistochemistry of tumor sections demonstrates similar results. (O) 81% of Olig2 and (P) 100% of Gfap cells in tumors expressed the CGNP marker Pax6.

To establish Olig2 progenitor contributions during CGNP development, we constructed a multi-functional mouse line by inserting tva, an avian-specific retroviral receptor, and an IRES-cre recombinase cassette into the endogenous Olig2 locus by homologous recombination (Figure 2A). When Olig2-tva-cre mice were crossed to ROSA26-eYFPFl/Fl conditional reporter mice, we found that 98% of YFP cells in the P7 cerebellar white matter (WM) expressed Olig2, consistent with oligodendroglial identity (Figure 2B, C, yellow arrows). At P21 most of these cells co-expressed myelin basic protein, and we observed small numbers of YFP PN and IN (Supp. Figure 1E, F). At P7, we detected a small subpopulation of YFP CGNP within the EGL, confined mainly to posterior cerebellar Lobes IX/X (Figure 2C-E, white arrows). At P21, we observed fate mapping to GC within the IGL (Figure 2F-I). Interestingly, only 3% of YFP cells expressed the GC marker Zic in the Lobe II IGL, but 58% of YFP cells were double positive in Lobe X (Figure 2G and I), indicating that Olig2 contributions to GC of the IGL are restricted mainly to the posterior lobes.

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A subset of cerebellar granule neurons derive from Olig2 progenitors

(A)Olig2-tva-cre allele and genotyping of the respective mutant mice using PCR to generate wild type 429bp and mutant (cre) 446bp products. (B-I) Fate mapping of Olig2-expressing cells with ROSA26-eYFPFl/Fl reporter. Co-localization of YFP and Olig2 proteins in sagittal sections of P7 cerebella reveals that YFP is expressed in oligodendrocytes within the cerebellar WM (yellow arrows in C) as well as in occasional cells within the EGL (white arrows). YFP-Pax6 labeling confirms the granule lineage character of YFP-expressing EGL cells (D, E, arrows). The IGL of P21 cerebella demonstrates lobe-specific differences in Olig2 fate mapping. (G) The IGL of Lobe II contains YFP cells that predominantly co-express Olig2 (arrow and cell shown, inset) but not the GC marker Zic. In contrast, the IGL of Lobe X (H) contains numerous YFP/Zic double-positive cells (arrow and inset). (I) Mean percentage (+/-SEM) of Olig2YFP and Zic1YFP double-positive cells in the IGL of lobe II and X. (J-M) In utero fate mapping of Olig2-expressing cells by RCAS-GFP virus injection into the rRL of E15.5 Olig2-tva-cre mice. Analysis of the CB at P7 reveals GFP to be expressed in Olig2 (K) WM cells, as well as Pax6 (L) and Zic (M) cells of the IGL in Lobe X.

We found Olig2 cells in the rRL from E14.5-18.5 (Figure 1 and data not shown). To test whether these cells in particular were a source of lobe IX-X CGNP, we injected the rRL of E15.5 Olig2-tva-cre animals with an avian retrovirus (RCAS-GFP), which can only transduce tva-expressing cells (Figure 2J). Fate mapping results analyzed at P7 indicated that infected rRL Olig2-tva cells generate Pax6 CGNP of the EGL and Zic GC within the IGL (Figure 2K-M, Suppl. Figure 1) of lobes IX and X. Taken together, these data indicate that Olig2 progenitors of the rRL make restricted contributions to the GC lineage, particularly in posterior lobes of the CB.

Smoothened activation within CNS hGFAP and Olig2 progenitors produces only medulloblastoma

To determine the significance of hGFAP- and Olig2-expressing progenitor populations for CNS tumorigenesis, we used hGFAP-cre and Olig2-tva-cre drivers, respectively, to conditionally express an activated Smo (SmoM2) allele fused with yellow fluorescent protein (YFP) sequences (Mao et al., 2006). All hGFAP-cre:SmoM2 mice developed diffuse tumors with the characteristic “small round blue cell” histology of MB (Figure 3A-D, Table 1). Olig2-tva-cre:SmoM2 mice developed tumors with similar histology, except that they were focal, localized to the posterior-lateral lobes and had significantly (p <0.01) later mortality (Figure 3E-H, Table 1), which was likely a result of their focal nature and the small numbers of CGNP targeted by Olig2-tva-cre.

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Development of medulloblastoma but not glioma from hGFAP and Olig2 precursors

Targeting hGFAP and Olig2 precursor cells with SmoM2 results in MB (A-H). While neoplastic transformation of hGFAP cells affects the entire cerebellar cortex (A-D), MBs from Olig2-expressing precursors are restricted to posterior cerebellar regions (E-H). Although hGFAP-cre and Olig2-cre are broadly expressed in all parts of the CNS, neoplastic lesions due to SmoM2 are restricted to the CB. Forebrain sections including the subventricular zone (SVZ) in adult animals appear normal (I-L). Transplantation of Olig2-tva-cre:SmoM2 tumor cells into SCID mice gives rise tumors both in hindbrain (M,N) and forebrain (O,P) regions. A, E and I-P are frontal, B-D and F-H are sagittal sections stained with H&amp;E. C and D are high power magnifications from B, so are G and H from F. Asterisks mark lateral ventricles in I, K and O and the IV ventricle in M. BS, brainstem.

Although hGFAP and Olig2 progenitor populations give rise, collectively, to CGNP as well as IN, astrocytes (AS) and oligodendrocytes (OL), we only observed cerebellar tumors with histological features of MB. Additionally, activated Hh signaling in cerebellar PN driven by Shh-creERT2 never resulted in MB (Table 1). These data demonstrate that hGFAP and Olig2 multipotent progenitor populations can produce MB.

Hh signaling can have mitogenic effects in spinal cord and forebrain (Mao et al., 2006; Rowitch et al., 1999; Fuccillo et al., 2006), and roles in forebrain glioma have been reported (Clement et al., 2007; Ehtesham et al., 2007). However, no evidence of gliomas or other primitive neuroectodermal tumors (PNET) in the forebrain was observed despite forced expression of SmoM2 in the subventricular zone, in NG2 progenitors, and in oligodendrocyte precursor cells (OPC) of the forebrain (Table 1, Figure 3I-L). Together, these data indicate a hindbrain- as well as a CGNP/lineage-restricted oncogenic response to Hh signaling, and raised the question of whether this was determined by the particular environmental niche of the EGL. To assess this, we transplanted freshly dissociated cells from Olig2-tva-cre:SmoM2 tumors into the forebrains and cerebella of SCID mice. Because tumors formed equally well in both locations (Figure 3M-P, Table 1), suggesting tumor propagation is governed primarily by cell-intrinsic factors rather than environment.

Unipotent CGNP are competent to produce medulloblastoma

In myeloid oncogenesis, multipotent cells rather than more restricted progenitor cells are thought to be the origin of leukemia stem cells (Faber and Armstrong, 2007). To test whether a restricted neuronal precursor can also give rise to MB, we activated SmoM2 in committed CGNP. Math1-cre drives expression within CGNP of the rRL and the entire EGL until P7-10, but not in PN, AS or IN (Machold and Fishell, 2005; Wang et al., 2005). In contrast, expression of the transcription factor Tlx3 (Hox11L2) is only seen in the EGL of Lobes VI-IX, but never in other lobes or the rRL (Suppl. Figure 2). Transgenic Math1-cre mice were generated using regulatory sequences 4 kb upstream of Math1 (Matei et al., 2005; Schüller et al., 2007) and Tlx3-cre mice were generated by gene targeting of Tlx3 (Xu et al., 2008). Fate mapping crosses involving a conditional ROSA26-eYFPFl/Fl reporter confirmed that Math1 and Tlx3 precursors contributed solely to the GC lineage (Figure 4A-H and data not shown).

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Late stage unipotent CGNP are competent to produce medulloblastoma

(A-H) Cre expression driven by Math1 or Tlx3 promoter sequences is normally restricted to granule lineage cell as shown by crosses with Rosa26-eYFP conditional reporter mice. (C, D, G) GC fate-mapped by Tlx3-cre are restricted to Lobes VI-IX. (F, H) In contrast, Calbindin-expressing PN do not derive from Math1 or Tlx3 precursors. (I-L) Both Math1 and Tlx3 granule neurons give rise to MB after activation of SmoM2, and Tlx3-cre driven tumors obeyed posterior restriction consistent with fate mapping (compare arrows in C, K).

When crossed with SmoM2Fl/Fl mice, all Math1-cre animals developed MBs that could be transplanted into the cerebella of SCID mice (Figure 4I, J, Table 1, also see (Yang et al., 2008)). Tlx3-cre:SmoM2 animals developed tumors in a posterior distribution in accord with the Tlx3 expression domain (Figure 4K, L). These data show that unipotent CGNP are capable of tumor formation when targeted with oncogenic SmoM2. The Shh transcriptional target, Gli1, is expressed in proliferating CGNP (Wechsler-Reya and Scott, 1999) and we could induce MB formation in Gli1-creERT2:SmoM2 mice until P14, but not at later postnatal stages.

Medulloblastomas exhibit a common phenotypic endpoint despite diverse cellular origins

To determine whether significant cellular (i.e. glial and neuronal diversity) and gene expression differences might exist in tumors derived from multipotent (e.g. hGFAP-cre) versus unipotent (e.g. Math1-cre) progenitor populations we performed Affymetrix gene expression profiling analyses, previously demonstrated to aid in distinguishing MB from other histologically similar primitive neuroectodermal tumors (Pomeroy et al., 2002). Comparison of expression profiles of the different murine tumors and Ptc medulloblastoma using principal component (PC) analysis confirmed that all tumors had a gene expression signature of medulloblastomas and were highly related to each other (Figure 5A). Along the PC1 coordinate — the direction of greatest transcriptomic sample variance in cerebellar development —centroids of the MB tumor profiles were significantly different from matched normal samples (p < 10). However, PC1 coordinate centroids were not significantly different for tumors derived from early (i.e., hGFAP- or Olig2-cre) versus late/unipotent (i.e., Math1- or Tlx3-cre) GC lineage progenitors (Suppl. Figure 3). In fact, only a very small number of genes (∼150 of 54,675 probes tested) were found to be uniquely and differentially expressed in each tumor sub-group. In line with this, extensive histological (Figures 3C, 3G, 4J, 4L) and immunohistochemical analysis (Figure 5B) failed to reveal any clear differences in the cellular composition of the tumors. Furthermore, hierarchical cluster analysis based on the 150 genes differentially expressed amongst the tumors did not show strict clustering of tumors based on cell of origin (Suppl. Figure 4). Together, these findings indicate that tumors derived from early (i.e., hGFAP-cre or Olig2-cre) or late/unipotent (i.e., Math1-cre or Tlx3-cre) GC lineage progenitors arrive at a common phenotypic endpoint.

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Medulloblastoma generated from diverse progenitor cell origins leads to a convergent phenotypic endpoint

(A) Principal component analysis of global gene expression in conditional MB models [Math1(green)-, Tlx3(magenta)-, hGFAP(red)-, Olig2(blue)-cre:SmoM2 and Ptc (turquoise)] and samples from normal littermate control CB (black circles) are mapped onto normal cerebellar developmental space as defined by a 7000 member, rank-normalized gene set (Kho et al., 2004), from developing CB at stages P1, 3, 5, 7, 10, 15, 21, 30, 50 and 60 (orange numbers). Tumors were related to early rather than late developmental stages of the CB as we have reported for human MB (Kho et al., 2004). (B) Immunohistochemical analysis confirms all murine tumors exhibit a similar immunohistochemical staining pattern to each other as well as human MB with respect to standard neuronal and glial neuropathological markers. Results are representative of at least two tumor samples.

Cells expressing Gfap and Olig2 in Hh-activated medulloblastomas are neoplastic

All mouse tumors analyzed showed histological features in common with some human MBs including expression of CGNP markers (Math1, Zic, Pax6), Gfap, a marker usually found to be expressed in astrocytes, and Olig2, which is required for OL development (Lu et al., 2002) (Figure 5C). Clinical symptoms of tumor burden in Math1-cre:SmoM2 tumors developed at an average age of 41 days (Table 1), however we found evidence of hyperplasia in the EGL at P0 and more clearly at P7 (Figure 6A, C). Strikingly, high expression of Olig2 was also found in these regions (Fig 6B, D). At P7, when the EGL is clearly discerned, excessive numbers of Olig2 cells were observed in the EGL but not RL of Math1-cre:SmoM2 mice (Figure 6D). Indeed, although Olig2 cells that co-expressed Pax6 (Figure 1I) or a Math1 proxy marker (Figure 6E) could be observed at antenatal stages until E18, such co-labeled cells were not detectable at P7 (Figure 6F) or later stages. Thus, persistent Olig2 expression in the EGL of Math1-cre:SmoM2 mice represents an early molecular marker of a pre-neoplastic state.

We found that only about 2% of mitotically active phosphoH3 (pH3) cells were Olig2 in fully developed Math1-cre:SmoM2 tumors (Figure 6G). Indeed, the vast majority of proliferating cells were Cre (Figure 6H), a proxy for Math1 expression, indicating that most cells contributing to MB growth are CGNP-like. In human tumors it is controversial whether cells expressing GFAP (or OLIG2) comprise tumor, or alternatively, represent intermingled cells from the normal brain. This is a critical point given that glial differentiation within MB would provide further evidence for a multipotent stem cell-like progenitor. To establish the nature of cells comprising MB, we dissociated Math1-cre:SmoM2 tumors and found that the SmoM2-YFP fusion protein, a marker of cells bearing the tumorigenic mutation, co-localized in cells expressing either Pax6, NeuN, Gfap or Olig2. Quantification revealed that YFP proteins co-localized with most if not all Pax6, Olig2 and Gfap (100%, 90%, 96%, respectively) cells in acutely dissociated tumors (Figure 6I-L). In addition, we identified Olig2 and Gfap cells that co-expressed the YFP reporter in vivo (Figure 6M, N). Thus, Olig2- and Gfap cells are tumor cells rather than infiltrated/normal glial cells.

The Gfap and Olig2 cells might represent simple astroglial and oligodendroglial tumor components. However, examination of Gfap and Olig2 cells of the cerebellar anlagen (Figure 1) suggested that such tumor cells could, alternatively, retain features of primitive CGNP. In support of the latter possibility, we found that 81% of Olig2 cells expressed Pax6 and that 100% of Gfap cells expressed Pax6 in acutely disassociated Math1-cre:SmoM2 tumors in vitro (Figure 6O, P). These findings indicate that cells with an immature/progenitor phenotype are present in Hh-activated MB.

Human medulloblastoma contain rare OLIG2 cells with progenitor features

In order to determine the significance our findings might have for human MB, we examined OLIG2 cell populations within a cohort of human MBs. Although our original findings in mice were all in the setting of Shh/Smo pathway activation, we found that OLIG2 cells were present in both classic (40%) as well as desmoplastic (Shh-related) (77%) subtypes of human MBs (Fig 7A-D; Suppl. Table 1), and comprised less than 5% of cells in all tumors. Fluorescent in situ hybridization (FISH) analysis showed that OLIG2 cells in human MB contained copy number gain at the C-MYC locus, confirming them as tumor cells and arguing against the possibility that they might represent intermingled normal cells (Figure 7E, F). As further support for OLIG2 cells being tumor cells we identified co-expression of OLIG2 and the terminal neuronal differentiation marker, NEUN, in rare cells within human tumors (Figure 7G), and we found rare OLIG2 cells that expressed the proliferation marker, Ki67 (Figure 7H). Together, these data suggest the possibility that some OLIG2 cells are tumor cells with immature features and may represent a type of MB progenitor cell.

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OLIG2 is expressed in human medulloblastoma

(A-D) OLIG2 expression in human classic and desmoplastic MB and percentage of tumors of each class that contain OLIG cells (also see Suppl. Tab 1). (E, F) Combined FISH and immunohistochemical analysis reveals the presence of tumor specific C-MYC genomic aberrations (Herms et al., 2000) within OLIG2 cells (yellow nuclei) of human MB (n=2). C-MYC aberrations were identical in OLIG2 and OLIG2 tumor cells. Note combined copy number gain (red signals, 5′ C-MYC) and structural aberration (loss of green signals, 3′ C-MYC), as detected using break apart probe set. Normal MYC loci show merged colors as (focal yellow signals). (G, H) OLIG2 co-localizes with NEUN, and the proliferation marker Ki67. (I) Scheme for experiments using RCAS-GFP infection of Olig2-tva-cre:SmoM2 tumor cells. (J-M) Double-fluorescence images show expression of GFP that co-localizes with immunoreactivity using antibodies against Olig2 and GC lineage markers. Note that while Olig2-tva-cre:SmoM2 express YFP the signal is very weak/undetectable compared with GFP encoded by RCAS virus, as shown (J-M). (N) Summary of findings at Day 6 in GFP-labeled cells. (O-Q) Analysis of acutely disassociated Olig2-tva-cre:SmoM2 tumor (Day 0). Of Olig2+ cells, 66% co-labeled with Pax6 and 12% with NeuN, such cells represented 4% and 1% of total cells in the tumor, respectively.

To mechanistically assess the lineage differentiation potential of Olig2 cells in the context of MB, we acutely disassociated tumors from Olig2-tva-cre:SmoM2 mice and infected these cultures with RCAS-GFP viruses, to perform fate mapping of tumor stem/progenitor cells (Figure 7I). In our experimental paradigm, cells must be Olig2-tva for infection with RCAS-GFP to occur. After six days in culture, we found that cells derived from Olig2 progenitors expressed Pax6, Zic or NeuN (Figure 7K-N). Significantly, of the 561 GFP cells counted, only 68 (12%) expressed Olig2 on Day 6 (Figure 7J, N), indicating differentiation along GC lineage axis with down-regulation of Olig2. Upon acute harvest and disassociation of tumors (Day 0), we found that only 8% expressed Olig2. Of these, 66% co-expressed Pax6 and 12% expressed NeuN (Figure 7O-Q). Contrasting this, on Day 6 in the tva fate map experiment, 52% of Olig2-derived progeny expressed NeuN. These data are consistent with the proposal that Olig2Pax6 cells lose Olig2 expression and progress to express the more mature markers such as Zic and NeuN during the 6 days in culture. We conclude that Olig2 cells in Olig2-tva-cre:SmoM2 tumors have the potential to produce GC-like progeny therefore supporting the possibility that they represent a subset of MB progenitor cells.

Discussion

Here we show that unipotent CGNP derive from multilineage embryonic CNS progenitors and that both populations are capable of generating MB when targeted with oncogenic SmoM2. Despite diverse origins, resulting tumors were similar leading to the conclusion that acquisition of CGNP identity is a critical determinant of progenitor cell competence to form Hedgehog-induced MB. These findings provide insight into MB origins with implications for development of targeted cellular therapies.

Olig2+ progenitors of RL give rise to a subset of granule cell neurons

By creating a knock-in transgenic mouse line, Olig2-tva-cre, we targeted Olig2 cells using two fate map approaches. Olig2-tva-cre intercrossed with conditional reporters (e.g., ROSA26-YFP) mapped a subset of GC in the EGL and IGL of posterior CB. We then confirmed that these cells are derived from Olig2 progenitors in the rRL by in utero infection of Olig2-tva-cre mice with an avian RCAS retrovirus reporter. Although we cannot exclude that Olig2 cells outside of rRL could contribute to CGNP, this possibility seems unlikely given previous work that has established the rRL as the source of the GC lineage. These data indicate that rRL Olig2 progenitors give rise to a subset of GC (Figure 8A), which might contribute to functional heterogeneity (Zong et al., 2005).

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Tumor competent cells of the CGNP lineage can give rise to diffuse and focal forms of medulloblastoma

(A) Scheme showing P7 neuronal progeny deriving from hGFAP- and Olig2-expressing progenitors of the embryonic CB. hGFAP cells produce the majority of granule cell precursors (GNP) as well as astrocytes (Glia) and IN. While some Olig2 cells derive from hGFAP cells (black dashed arrow), additional sources are possible. Olig2 cells of the rRL produce a subset of GNP in cerebellar lobes IX and X, and also give rise to IN and oligodendrocytes (Glia) in all lobes (red arrows). The blue dashed arrow indicates the possibility of GNP de-differentiation in the context of Hh-induced MB. (B) Activation of SmoM2 in GNP using hGFAP- or Math1-cre produced diffuse MB involving most cerebellar lobes and the vermis, while focal, posterior-lateral MB was produced from the Olig2-cre driver; Tlx3-cre produced tumors with posterior restriction. (C) Possible cellular pathways of tumorigenesis. Introduction of an oncogene (red arrowheads) into multipotent (A, giving rise to distinct progeny types B1, B2, B3), unipotent (C2) or even differentiated (D2) subtypes of a tumor-competent lineage (e.g., GNP of CB) might result in a uniform tumor phenotype providing only one stage (C2) is capable of rapid expansion in response to the signal. This seems the simplest model to fit our collective findings; however, tumor propagation from an A or B2 cell cannot be ruled out. The later possibility of de-differentiation from D2 > C2 is speculative and not supported by the present work.

Oncogenic signaling and lineage-restricted factors cooperate to determine tumor competence in CNS progenitors

Human inherited cancer syndromes show tumor formation in only a subset of cells within specific lineages despite germline transmission of tumor promoting mutations (e.g., p53, pRB-loss-of-function). Although several reports indicate that the Hh pathway is active in low- and high-grade gliomas (Ehtesham et al., 2007; Clement et al., 2007), in human Goltz-Gorlin syndrome, inherited heterozygous mutation of PTCH or SUFU lead to MB, but not gliomas. We failed to detect gliomas despite robust SmoM2 activation in forebrain progenitors, including developing neural stem cells and persistent progenitors of the SVZ and cortex expressing GFAP (Doetsch et al., 1999), NG2/Olig2 (Ligon et al., 2006) and Gli1 (Machold et al., 2003; Ahn and Joyner, 2005). Further work is needed to establish whether additional mutations (e.g., loss of p53) in concert with Hh activation might be necessary to enable glioma formation. Another possibility is that Shh activation biases towards neuronal lineage versus glial differentiation and therefore a MB phenotype and not glial lineages or glial tumor phenotype. Although we cannot rule this out, we think this explanation is unlikely for the following reasons. First, assuming that Shh promotes neuronal tumorigenesis, then one might expect to see development of such tumors at other CNS sites (e.g. supratentorial PNETs); this is not observed in human Goltz-Gorlin syndrome patients or in the mouse models we have studied. Second, we did not see other neuronal tumor types in the CB despite activation of Shh in PN and IN (Table 1). Third, we have recently shown that SmoM2 activation in the dentate gyrus causes hypertrophy but not cancer (Han et al., 2008).

Acquisition of granule cell lineage identity is critical for Hh-induced medulloblastoma formation

To identify progenitors competent for MB formation, we activated SmoM2 in a spectrum of stage- and position-specific progenitors during CGNP development. Both multipotent (e.g., hGFAP and Olig2) progenitor populations within the cerebellar anlage as well as unipotent/restricted CGNP readily gave rise to MB with 100% penetrance. Despite these diverse origins, all SmoM2-activated MBs we analyzed exhibited a strikingly similar tumor phenotype at histological, immunohistochemical and expression profiling levels. The common final tumor phenotype is consistent with (1) a common cell or origin for all tumors, or (2) initial origins from any stem/progenitor which later converge on an amplifying CGNP-like tumor cell that dominates the ultimate phenotype (see Figure 8C). In either case, these findings support the conclusion that acquisition of CGNP identity is important for Hh-induced MB formation. Because orthotopically transplanted Olig2-tva-cre:SmoM2 tumors were able to grow within the forebrain and CB, restriction of tumor susceptibility is evidently governed by cell-intrinsic factors. Further work is needed to identify the underlying molecular determinants of CGNP “tumor competence” that lead to MB formation.

Hh-activated medulloblastoma can be generated in the EGL independent of VZ contributions

Human desmoplastic and classic MB have been suggested to arise from CGNP EGL (Schüller et al., 2005; Bühren et al., 2000) or progenitors of the VZ (Salsano et al., 2007; Katsetos et al., 1995), respectively. Our studies demonstrate that Hh-induced MB can be initiated by precise restriction of activating mutations to the EGL using Tlx3-cre:SmoM2 and postnatally-induced Gli1-creERT2:SmoM2 without activation of SmoM2 in the less differentiated cell populations of the rRL or VZ. Furthermore, while the time of tumor onset and the degree of tumor distribution varied depending on the cre drivers used (Table 1, Figure 8B), these variations appear to be based on the number/extent/ location of the total CGNP population targeted (i.e., broad diffuse versus small lateral populations). In addition, tumor masses never developed in the VZ or rRL proper, suggesting that further work will be needed to determine whether additional oncogenic pathways (e.g. MYC, Wnt) might lead to MB formation in these regions.

Evidence that Gfap+ and Olig2+ cells in medulloblastomas are neoplastic

Our data highlight that both mouse and human MBs exhibit extensive cellular heterogeneity. GFAP cells are commonly identified in human MB (Schüller et al., 2004) and the morphology of those cells ranges from small tumor cells with a relatively large nucleus and a sparse, ring-like cytoplasm to cells with a rather differentiated phenotype and branched processes. We observed that OLIG2 cells, present in human MB, also varied in size and morphology. Using MYC FISH in human MB and YFP tracer expression in murine MB we demonstrate that OLIG2 and GFAP cells are bona fide tumor cells and not just infiltrating cells arising from the normal surrounding brain. In addition we found small numbers of proliferating Olig2 and Gfap cells within mouse and to a lesser degree human MB as further evidence that these heterogeneous elements represent tumor cells.

In human gliomas OLIG2 is expressed in tumor stem/progenitor cells and maintains the proliferation competence of both normal and tumorigenic progenitors of the murine forebrain (Ligon et al., 2007). However, given our data that OLIG2 is only rarely expressed in proliferating cells and is only present in 40% of classic and 77% of desmoplastic human MB cases, it seems clear that MB tumor growth is largely driven by rapidly dividing progenitor cells with CGNP characteristics, rather than OLIG2 cells. Might GFAP and OLIG2 cells in MBs have other roles as relatively quiescent tumor progenitor cells? Our in vitro tumor fate mapping demonstrates that Hh-transformed Olig2 cells from murine MB can produce GC lineage-like progeny with advanced differentiation measured by NeuN labeling. Whether Olig2 tumor cells from other mouse models (i.e., non-Olig2-cre derived) have this capability remains to be determined.

Our data suggest that GFAP and OLIG2 expression in MB is not associated with “differentiated” astrocytes and oligodendrocytes. While almost all Gfap and Olig2 cells in Hh-activated murine MB co-expressed Pax6 and other CGNP markers, the “biphenotypic” (e.g., OLIG2-NEUN) staining pattern is unlikely to be a specific consequence of Hh signaling, because we also observed this phenomenon in classic MBs, which are not associated with Hh mutations (Pietsch et al., 1997). We suggest that GFAP and OLIG2 label cells with a phenotype reminiscent of radial glia and less differentiated CGNP progenitors of the rRL (Figure 8A).

Does medulloblastoma arise from a multipotent stem cell?

MB was one of the first solid tumors for which “cancer stem cells” were reported (Hemmati et al., 2003). Cancer stem cells are classically described to be a tumor subpopulation with (1) unlimited capacity for self-renewal and the ability to regenerate tumor upon transplantation, and (2) the potential to produce multilineage progeny similar to distinct cell types of a cognate organ (Reya et al., 2001). The question of authentic glial cell production has critical implications for understanding MB progenitors and their characterization as multipotent cancer stem cells. However, evidence for multilineage differentiation of MB stem cells has been based on the expression of relatively few neuronal and glial cell markers with uncertain relation to either differentiated cell types of the CB or CNS multipotent stem cells. For example, the commonly used marker, GFAP, marks Type B stem cells of the subventricular zone (Doetsch et al., 1999). We show that Gfap and Olig2 mark cells in MBs reminiscent of those found during early CGNP development, but not differentiated glia. These findings argue against existence of MB progenitors with multilineage (neuronal and glial) contributions to the tumor mass. However, they are consistent with the possibility that MB could originate from either a multipotent or unipotent progenitor (Figure 8C).

We used lineage tracing to clarify the possible relationship between cells found during GC lineage development and those that are tumor-competent to produce MB. Our findings highlight distinctions between “cell-of-origin” (that acquires the oncogenic mutation) and “tumor-propagating” cell, responsible for major growth and phenotypic characteristics of the ultimate tumor. For instance, if unipotent cell identity (“Type C2”) is necessary for tumor growth (Figure 8C), then a tumor arising from mutation of a Type A or Type C2 cell would appear identical notwithstanding their different cells-of-origin. This also raises the possibility of a “de-differentiation” pathway to produce tumors with mixed multipotent and unipotent features, as has been suggested for certain leukemia stem cells (Krivtsov et al., 2006). Although our data do not provide direct evidence for de-differentiation, tumors from postnatally-induced Gli1-creERT2:SmoM2 (J. Mao, A. McMahon and K. Ligon, unpublished observations) and Math1-creERT2:PtcFl/Fl animals (Z. J. Yang and R. J. Wechsler-Reya, unpublished observations) contain many Olig2 cells, consistent with the possibility that late-stage CGNP can adopt less-differentiated characteristics (Figure 8). Further investigation is needed to determine whether dedifferentiation of mature granule cells (D2 >C2) constitutes a biologically feasible or clinically relevant pathway of tumor formation.

Olig2+ progenitors of RL give rise to a subset of granule cell neurons

By creating a knock-in transgenic mouse line, Olig2-tva-cre, we targeted Olig2 cells using two fate map approaches. Olig2-tva-cre intercrossed with conditional reporters (e.g., ROSA26-YFP) mapped a subset of GC in the EGL and IGL of posterior CB. We then confirmed that these cells are derived from Olig2 progenitors in the rRL by in utero infection of Olig2-tva-cre mice with an avian RCAS retrovirus reporter. Although we cannot exclude that Olig2 cells outside of rRL could contribute to CGNP, this possibility seems unlikely given previous work that has established the rRL as the source of the GC lineage. These data indicate that rRL Olig2 progenitors give rise to a subset of GC (Figure 8A), which might contribute to functional heterogeneity (Zong et al., 2005).

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Tumor competent cells of the CGNP lineage can give rise to diffuse and focal forms of medulloblastoma

(A) Scheme showing P7 neuronal progeny deriving from hGFAP- and Olig2-expressing progenitors of the embryonic CB. hGFAP cells produce the majority of granule cell precursors (GNP) as well as astrocytes (Glia) and IN. While some Olig2 cells derive from hGFAP cells (black dashed arrow), additional sources are possible. Olig2 cells of the rRL produce a subset of GNP in cerebellar lobes IX and X, and also give rise to IN and oligodendrocytes (Glia) in all lobes (red arrows). The blue dashed arrow indicates the possibility of GNP de-differentiation in the context of Hh-induced MB. (B) Activation of SmoM2 in GNP using hGFAP- or Math1-cre produced diffuse MB involving most cerebellar lobes and the vermis, while focal, posterior-lateral MB was produced from the Olig2-cre driver; Tlx3-cre produced tumors with posterior restriction. (C) Possible cellular pathways of tumorigenesis. Introduction of an oncogene (red arrowheads) into multipotent (A, giving rise to distinct progeny types B1, B2, B3), unipotent (C2) or even differentiated (D2) subtypes of a tumor-competent lineage (e.g., GNP of CB) might result in a uniform tumor phenotype providing only one stage (C2) is capable of rapid expansion in response to the signal. This seems the simplest model to fit our collective findings; however, tumor propagation from an A or B2 cell cannot be ruled out. The later possibility of de-differentiation from D2 > C2 is speculative and not supported by the present work.

Oncogenic signaling and lineage-restricted factors cooperate to determine tumor competence in CNS progenitors

Human inherited cancer syndromes show tumor formation in only a subset of cells within specific lineages despite germline transmission of tumor promoting mutations (e.g., p53, pRB-loss-of-function). Although several reports indicate that the Hh pathway is active in low- and high-grade gliomas (Ehtesham et al., 2007; Clement et al., 2007), in human Goltz-Gorlin syndrome, inherited heterozygous mutation of PTCH or SUFU lead to MB, but not gliomas. We failed to detect gliomas despite robust SmoM2 activation in forebrain progenitors, including developing neural stem cells and persistent progenitors of the SVZ and cortex expressing GFAP (Doetsch et al., 1999), NG2/Olig2 (Ligon et al., 2006) and Gli1 (Machold et al., 2003; Ahn and Joyner, 2005). Further work is needed to establish whether additional mutations (e.g., loss of p53) in concert with Hh activation might be necessary to enable glioma formation. Another possibility is that Shh activation biases towards neuronal lineage versus glial differentiation and therefore a MB phenotype and not glial lineages or glial tumor phenotype. Although we cannot rule this out, we think this explanation is unlikely for the following reasons. First, assuming that Shh promotes neuronal tumorigenesis, then one might expect to see development of such tumors at other CNS sites (e.g. supratentorial PNETs); this is not observed in human Goltz-Gorlin syndrome patients or in the mouse models we have studied. Second, we did not see other neuronal tumor types in the CB despite activation of Shh in PN and IN (Table 1). Third, we have recently shown that SmoM2 activation in the dentate gyrus causes hypertrophy but not cancer (Han et al., 2008).

Acquisition of granule cell lineage identity is critical for Hh-induced medulloblastoma formation

To identify progenitors competent for MB formation, we activated SmoM2 in a spectrum of stage- and position-specific progenitors during CGNP development. Both multipotent (e.g., hGFAP and Olig2) progenitor populations within the cerebellar anlage as well as unipotent/restricted CGNP readily gave rise to MB with 100% penetrance. Despite these diverse origins, all SmoM2-activated MBs we analyzed exhibited a strikingly similar tumor phenotype at histological, immunohistochemical and expression profiling levels. The common final tumor phenotype is consistent with (1) a common cell or origin for all tumors, or (2) initial origins from any stem/progenitor which later converge on an amplifying CGNP-like tumor cell that dominates the ultimate phenotype (see Figure 8C). In either case, these findings support the conclusion that acquisition of CGNP identity is important for Hh-induced MB formation. Because orthotopically transplanted Olig2-tva-cre:SmoM2 tumors were able to grow within the forebrain and CB, restriction of tumor susceptibility is evidently governed by cell-intrinsic factors. Further work is needed to identify the underlying molecular determinants of CGNP “tumor competence” that lead to MB formation.

Hh-activated medulloblastoma can be generated in the EGL independent of VZ contributions

Human desmoplastic and classic MB have been suggested to arise from CGNP EGL (Schüller et al., 2005; Bühren et al., 2000) or progenitors of the VZ (Salsano et al., 2007; Katsetos et al., 1995), respectively. Our studies demonstrate that Hh-induced MB can be initiated by precise restriction of activating mutations to the EGL using Tlx3-cre:SmoM2 and postnatally-induced Gli1-creERT2:SmoM2 without activation of SmoM2 in the less differentiated cell populations of the rRL or VZ. Furthermore, while the time of tumor onset and the degree of tumor distribution varied depending on the cre drivers used (Table 1, Figure 8B), these variations appear to be based on the number/extent/ location of the total CGNP population targeted (i.e., broad diffuse versus small lateral populations). In addition, tumor masses never developed in the VZ or rRL proper, suggesting that further work will be needed to determine whether additional oncogenic pathways (e.g. MYC, Wnt) might lead to MB formation in these regions.

Evidence that Gfap+ and Olig2+ cells in medulloblastomas are neoplastic

Our data highlight that both mouse and human MBs exhibit extensive cellular heterogeneity. GFAP cells are commonly identified in human MB (Schüller et al., 2004) and the morphology of those cells ranges from small tumor cells with a relatively large nucleus and a sparse, ring-like cytoplasm to cells with a rather differentiated phenotype and branched processes. We observed that OLIG2 cells, present in human MB, also varied in size and morphology. Using MYC FISH in human MB and YFP tracer expression in murine MB we demonstrate that OLIG2 and GFAP cells are bona fide tumor cells and not just infiltrating cells arising from the normal surrounding brain. In addition we found small numbers of proliferating Olig2 and Gfap cells within mouse and to a lesser degree human MB as further evidence that these heterogeneous elements represent tumor cells.

In human gliomas OLIG2 is expressed in tumor stem/progenitor cells and maintains the proliferation competence of both normal and tumorigenic progenitors of the murine forebrain (Ligon et al., 2007). However, given our data that OLIG2 is only rarely expressed in proliferating cells and is only present in 40% of classic and 77% of desmoplastic human MB cases, it seems clear that MB tumor growth is largely driven by rapidly dividing progenitor cells with CGNP characteristics, rather than OLIG2 cells. Might GFAP and OLIG2 cells in MBs have other roles as relatively quiescent tumor progenitor cells? Our in vitro tumor fate mapping demonstrates that Hh-transformed Olig2 cells from murine MB can produce GC lineage-like progeny with advanced differentiation measured by NeuN labeling. Whether Olig2 tumor cells from other mouse models (i.e., non-Olig2-cre derived) have this capability remains to be determined.

Our data suggest that GFAP and OLIG2 expression in MB is not associated with “differentiated” astrocytes and oligodendrocytes. While almost all Gfap and Olig2 cells in Hh-activated murine MB co-expressed Pax6 and other CGNP markers, the “biphenotypic” (e.g., OLIG2-NEUN) staining pattern is unlikely to be a specific consequence of Hh signaling, because we also observed this phenomenon in classic MBs, which are not associated with Hh mutations (Pietsch et al., 1997). We suggest that GFAP and OLIG2 label cells with a phenotype reminiscent of radial glia and less differentiated CGNP progenitors of the rRL (Figure 8A).

Does medulloblastoma arise from a multipotent stem cell?

MB was one of the first solid tumors for which “cancer stem cells” were reported (Hemmati et al., 2003). Cancer stem cells are classically described to be a tumor subpopulation with (1) unlimited capacity for self-renewal and the ability to regenerate tumor upon transplantation, and (2) the potential to produce multilineage progeny similar to distinct cell types of a cognate organ (Reya et al., 2001). The question of authentic glial cell production has critical implications for understanding MB progenitors and their characterization as multipotent cancer stem cells. However, evidence for multilineage differentiation of MB stem cells has been based on the expression of relatively few neuronal and glial cell markers with uncertain relation to either differentiated cell types of the CB or CNS multipotent stem cells. For example, the commonly used marker, GFAP, marks Type B stem cells of the subventricular zone (Doetsch et al., 1999). We show that Gfap and Olig2 mark cells in MBs reminiscent of those found during early CGNP development, but not differentiated glia. These findings argue against existence of MB progenitors with multilineage (neuronal and glial) contributions to the tumor mass. However, they are consistent with the possibility that MB could originate from either a multipotent or unipotent progenitor (Figure 8C).

We used lineage tracing to clarify the possible relationship between cells found during GC lineage development and those that are tumor-competent to produce MB. Our findings highlight distinctions between “cell-of-origin” (that acquires the oncogenic mutation) and “tumor-propagating” cell, responsible for major growth and phenotypic characteristics of the ultimate tumor. For instance, if unipotent cell identity (“Type C2”) is necessary for tumor growth (Figure 8C), then a tumor arising from mutation of a Type A or Type C2 cell would appear identical notwithstanding their different cells-of-origin. This also raises the possibility of a “de-differentiation” pathway to produce tumors with mixed multipotent and unipotent features, as has been suggested for certain leukemia stem cells (Krivtsov et al., 2006). Although our data do not provide direct evidence for de-differentiation, tumors from postnatally-induced Gli1-creERT2:SmoM2 (J. Mao, A. McMahon and K. Ligon, unpublished observations) and Math1-creERT2:PtcFl/Fl animals (Z. J. Yang and R. J. Wechsler-Reya, unpublished observations) contain many Olig2 cells, consistent with the possibility that late-stage CGNP can adopt less-differentiated characteristics (Figure 8). Further investigation is needed to determine whether dedifferentiation of mature granule cells (D2 >C2) constitutes a biologically feasible or clinically relevant pathway of tumor formation.

Experimental Procedures

Animal Procedures

For fate mapping, various cre-strains (Table 1) were intercrossed with ROSA26-eYFPFl/Fl or CAG-CAT-eGFPFl/Fl conditional reporter mice. At least three cerebella were harvested for each different time point fixed overnight in 4% paraformaldehyde/PBS. For tumor generation, cre-strains were intercrossed with SmoM2Fl/Fl mice and followed for survival and subsequent Kaplan-Meier analysis. For injections into forebrains or cerebella of SCID mice, tumor cells were freshly dissociated and resuspended in 1 μl PBS at a concentration of 10/μl (see Suppl. Methods). See Supplemental Methods for detailed information on generation, acquisition and genotyping of transgenic mouse strains and procedures including RCAS infection of Olig2-tva-cre embryonic hindbrain. All animal procedures were approved by the Institutional Animal Care and Use Committees at DFCI and UCSF.

Tissue collection, histological analysis, immunohistochemistry (IHC), in situ hybidization and photomicroscopy

Human tissue samples were obtained in accordance with the rules and regulations for human tissue collection; use in this study was approved by the Institutional Review Boards of the Brigham and Women’s Hospital/Partners Healthcare and Children’s Hospital Boston. Tissue samples were processed using standard techniques. Detailed protocols for in situ hybridization and immunohistochemistry, immuno-FISH as well as all antibodies used in this study are listed in Supplemental Methods and are available on request. Photomicrographs were taken with Zeiss Axioskop/Axiocam imaging or Nikon E600/SPOT imaging systems. For whole mount pictures of mouse MBs, fluorescent and bright-field images were overlaid using Photoshop software. A Leica TCS SL system was used for confocal microscopy.

Cell culture

Cultures from Math1-cre:SmoM2 tumors were established by triturating tumor tissue in DMEM medium and plating the cells on poly-D-Lysine coated dishes. Acutely dissociated Math1-cre, SmoM2 tumor cells were fixed 12 hours after plating. Long-term cultures were analyzed after 4 weeks in culture and 3 passages. Immunostaining was performed as described above. Cells from Olig2-tva-cre:SmoM2 tumors were infected with virus carrying RCAS(A)-GFP (2×10 cfu/ml; gift of C. Cepko, Harvard Medical School) after 24 hr culture in serum-free medium. Serum-containing medium was then added; after a total culture time of 6 days, cells were fixed and stained.

Statistics and quantification

For in vivo fate mapping in Olig2-tva-cre mice, counts were derived from analysis of 3 independent animals, and at least 10 20X fields of labeled sections of CB focused around Lobe X. For cell culture counts, cells were derived from pups of at least four separate litters. Each litter yielded material for two six-well plates of cerebellar and one six-well plate of ventricular cultures. Three wells of a six-well plate were used for each treatment (EGF, Shh, vehicle). At least 100 cells were assessed in each sample to determine the fraction of Olig2, Zic or Gfap cells. Statistical analysis was performed using the unpaired Student’s t-test with a two-tailed p-value.

Microarray Data and Analysis

The transcriptome of 21 cre-activated tumors and control cerebellar tissues were profiled using the Affymetrix GeneChip Mouse Genome 430 2.0 Plus [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc={"type":"entrez-geo","attrs":{"text":"GSE11859","term_id":"11859"}}GSE11859]. Transcriptomes of the developing mouse CB P1-60 were profiled using the Affymetrix Murine Mu11K set as described previously (Kho et al., 2004) and principal component analysis (PCA) was used to determine the global similarity and variation between the transcriptome profiles of the different tissues (Johnson and Wichern, 2002). For detailed description, see supplementary methods.

Animal Procedures

For fate mapping, various cre-strains (Table 1) were intercrossed with ROSA26-eYFPFl/Fl or CAG-CAT-eGFPFl/Fl conditional reporter mice. At least three cerebella were harvested for each different time point fixed overnight in 4% paraformaldehyde/PBS. For tumor generation, cre-strains were intercrossed with SmoM2Fl/Fl mice and followed for survival and subsequent Kaplan-Meier analysis. For injections into forebrains or cerebella of SCID mice, tumor cells were freshly dissociated and resuspended in 1 μl PBS at a concentration of 10/μl (see Suppl. Methods). See Supplemental Methods for detailed information on generation, acquisition and genotyping of transgenic mouse strains and procedures including RCAS infection of Olig2-tva-cre embryonic hindbrain. All animal procedures were approved by the Institutional Animal Care and Use Committees at DFCI and UCSF.

Tissue collection, histological analysis, immunohistochemistry (IHC), in situ hybidization and photomicroscopy

Human tissue samples were obtained in accordance with the rules and regulations for human tissue collection; use in this study was approved by the Institutional Review Boards of the Brigham and Women’s Hospital/Partners Healthcare and Children’s Hospital Boston. Tissue samples were processed using standard techniques. Detailed protocols for in situ hybridization and immunohistochemistry, immuno-FISH as well as all antibodies used in this study are listed in Supplemental Methods and are available on request. Photomicrographs were taken with Zeiss Axioskop/Axiocam imaging or Nikon E600/SPOT imaging systems. For whole mount pictures of mouse MBs, fluorescent and bright-field images were overlaid using Photoshop software. A Leica TCS SL system was used for confocal microscopy.

Cell culture

Cultures from Math1-cre:SmoM2 tumors were established by triturating tumor tissue in DMEM medium and plating the cells on poly-D-Lysine coated dishes. Acutely dissociated Math1-cre, SmoM2 tumor cells were fixed 12 hours after plating. Long-term cultures were analyzed after 4 weeks in culture and 3 passages. Immunostaining was performed as described above. Cells from Olig2-tva-cre:SmoM2 tumors were infected with virus carrying RCAS(A)-GFP (2×10 cfu/ml; gift of C. Cepko, Harvard Medical School) after 24 hr culture in serum-free medium. Serum-containing medium was then added; after a total culture time of 6 days, cells were fixed and stained.

Statistics and quantification

For in vivo fate mapping in Olig2-tva-cre mice, counts were derived from analysis of 3 independent animals, and at least 10 20X fields of labeled sections of CB focused around Lobe X. For cell culture counts, cells were derived from pups of at least four separate litters. Each litter yielded material for two six-well plates of cerebellar and one six-well plate of ventricular cultures. Three wells of a six-well plate were used for each treatment (EGF, Shh, vehicle). At least 100 cells were assessed in each sample to determine the fraction of Olig2, Zic or Gfap cells. Statistical analysis was performed using the unpaired Student’s t-test with a two-tailed p-value.

Microarray Data and Analysis

The transcriptome of 21 cre-activated tumors and control cerebellar tissues were profiled using the Affymetrix GeneChip Mouse Genome 430 2.0 Plus [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc={"type":"entrez-geo","attrs":{"text":"GSE11859","term_id":"11859"}}GSE11859]. Transcriptomes of the developing mouse CB P1-60 were profiled using the Affymetrix Murine Mu11K set as described previously (Kho et al., 2004) and principal component analysis (PCA) was used to determine the global similarity and variation between the transcriptome profiles of the different tissues (Johnson and Wichern, 2002). For detailed description, see supplementary methods.

Supplementary Material

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Acknowledgements

The authors are grateful to Rob Wechsler-Reya for sharing unpublished data, to Charles D. Stiles for comments and to Jason Ling, Sovann Kaing, Dong-in Yuk, Natalie Vena and Emily Learner for expert technical assistance. U.S. was supported by a fellowship from the Dr. Mildred-Scheel-Stiftung für Krebsforschung and by the Max-Eder-Nachwuchsgruppenprogramm of the Deutsche Krebshilfe. V.M.H. thanks the Netherlands Organization for Scientific Research (NWO) for a TALENT-stipend. J.M. was supported by a grant from the Charles King Trust Medical Foundation. Y-G. H., A.K.D. and E.H. thank the American Brain Tumor Foundation for support. This work was supported by grants from the NIH to K.L.L. (NS047213), A.K. (NS040828). A.P.M. (NS033642) and D.H.R. (NS047527), the James S. McDonnell Research Foundation (to D.H.R.), the March of Dimes Foundation (to D.H.R.), and a grant from the Pediatric Brain Tumor Foundation of the United States (to A.A-B. and D.H.R.). D.H.R is a HHMI Investigator. All authors declare that they have no competing financial interests.

Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115
Howard Hughes Medical Institute, UCSF, 513 Parnassus Avenue, San Francisco CA 94143
Institute for Regeneration Medicine, UCSF, 513 Parnassus Avenue, San Francisco CA 94143
Dept. of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138
Informatics Program, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115
Dept. of Neurological Surgery, UCSF, 505 Parnassus Avenue, San Francisco, CA 94143
Dept. of Pediatrics, UCSF, 505 Parnassus Avenue, San Francisco, CA 94143
Dept. of Pathology, Brigham and Women’s Hospital, 75 Francis Street
Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, and Dept. of Pathology, Children’s Hospital, Boston, MA 02115.
Center for Neuropathology, Ludwig-Maximilians-Universität, Feodor-Lynen-Strasse 23, 81377 Munich, Germany.
These authors contributed equally.
Authors for correspondence: ude.fscu.sdep@dhctiwor; ude.dravrah.icfd@nogil_htiek
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Abstract

Origins of the brain tumor, medulloblastoma, from stem cells or restricted progenitor cells are unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted CNS progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNP) derive from hGFAP and Olig2 RL progenitors. Hh activation in a spectrum of early and late stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and that they retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors.

Abstract

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