Gene expression profiling of human MBs reveals that they can be subdivided into four distinct subgroups, two of which manifest either SHH or WNT pathway upregulation (26
). In humans, less than a quarter of MBs manifest grossly aberrant SHH pathway activation, but most mouse models of MB, including those studied here, molecularly recapitulate the human SHH subgroup. Mouse MBs with a Shh signature appear to arise from GNPs in the postnatal cerebellum (3
). In contrast, several lines of evidence suggest that WNT-driven MBs are generated from more primitive embryonic neuronal progenitors (27
). The cells that initiate the other classes of human MBs have not been identified.
Shh drives the proliferation of GNPs during postnatal cerebellar development by inducing pro-proliferative genes that include Gli1
, the D-type cyclins Ccnd1
, and Mycn
). Similarly, Atoh1
expression correlates with Shh signaling and is specifically increased in mouse and human MBs that exhibit Shh pathway mutations (3
). However, despite the fact that Atoh1 expression is restricted to proliferating GNPs within the EGL and is essential for proper cerebellar histogenesis (8
is not a direct target of either Shh signaling or N-Myc in GNPs but functions in a parallel pathway (15
). Notably, enforced expression of either N-Myc (31
) or Atoh1 alone (this study) in primary GNPs is unable to induce medulloblastoma, although both genes collaborate with the Shh signaling pathway to accelerate tumor onset. A major difference between them is that enforced N-Myc expression in primary GNPs grown in the presence of Shh is unable to counter the effects of BMP4 (15
) while Atoh1 can. Unlike GNPs overexpressing Gli1, those forced to express Atoh1, although able to proliferate in the presence of Shh, were unable to proliferate in its absence. Instead, when Shh was transiently removed from the culture medium, Atoh1 overexpression maintained quiescent GNPs in a prolonged Shh-responsive state, thereby synergizing with re-added Shh to enhance subsequent mitogenesis. Conversely, acute deletion of Atoh1
in GNPs limited their response to Shh and accelerated neuronal differentiation.
When GNPs explanted from MB tumor-prone Ptch+/−
mice were transduced with vectors encoding either Gli1 or Atoh1 and transplanted into the brains of naïve animals, MB formation was more significantly accelerated by enforced Atoh1 expression. Consistent with the concept that Gli1 is a key arbiter of Shh signaling and that Atoh1 functions in a parallel pathway, MBs arising from Gli1-transduced donor cells did not inactivate the wild-type Ptch1
allele, whereas its expression was invariably lost in those MBs whose formation was enhanced by Atoh1. Expression of representative neuronal markers (Tuj1, Neuro D1, p27Kip1
, NeuN) was significantly reduced in response to Atoh1. Moreover, a detailed analysis of gene expression differences between MBs induced by overexpression of Gli1 and Atoh1 in this setting indicated that the majority of differentially regulated genes control processes of cell adhesion, morphogenesis, development, neurogenesis, and neuronal differentiation, but not cell proliferation or apoptosis. Notably, the levels of expression of prototypic cell cycle genes, including Ccnd1
, and Mycn
, each of which can accelerate MB formation in the Ptch1+/−
), were not significantly different in MBs that were accelerated by Atoh1 versus Gli1. Together, these findings argue that Atoh1 overexpression inhibits the further differentiation of GNPs, thereby extending and enhancing their response to Shh/Gli1 pathway activation.
Shh-induced proliferation is opposed by a parallel BMP2/4-mediated signaling pathway that accelerates exit of GNPs from the cell division cycle and induces their concomitant differentiation (19
). BMPs trigger the proteasomal degradation of Atoh1, but enforced expression of Atoh1 overrides these BMP-mediated effects (19
). To further investigate the mechanism by which Atoh1 antagonizes the differentiation-inducing effects of BMP2/4, we used a tamoxifen-inducible Atoh1-ER gene to override the effects of BMP4 on GNP gene expression and again applied microarray gene expression profiling to identify Atoh1-responsive genes. The protocol employed for this experiment minimized expression of the endogenous cellular Atoh1
gene and allowed a kinetic analysis of genes that responded rapidly to conditional Atoh1 induction. An unsupervised functional classification of Atoh1-ER-responsive genes that exhibited at least two-fold variations in expression identified 189 unique genes, 43 of which regulated cell differentiation and only 15 of which were annotated as ones controlling cell proliferation. Indeed, almost half of the latter were also categorized as governing differentiation processes. Included among these was a group of downregulated genes previously implicated in regulating BMP2/4 signaling, including Dlx1
), as well as genes such as Ntn1
and Slit that govern axon guidance (41
) and Cxcr4
, which encodes a G protein-coupled chemokine receptor that regulates GNP migration within the EGL (42
). Otx2, the expression of which is essential for proper cerebellar development and has been found to be overexpressed in human medulloblastoma cell lines (43
) was overexpressed in half of the MBs that arose spontaneously in tumor prone mice exhibiting Shh pathway activation; however, its expression was not significantly changed in Gli1/Atoh1 overexpressing tumors. Moreover, although Flora and collaborators recently reported that Atoh1 can directly induce Gli2 (9
), we failed to see an increase in Gli2 transcription after 4HT-mediated Atoh1-ER induction in primary GNPs. Pretreatment of cells with BMP prior to Atoh1 induction and/or variations in the timing of Gli2 induction in the different experimental settings might well account for the apparent discrepancies. We conclude that Atoh1 protein turnover and concomitant neuronal differentiation is not only accelerated in response to BMP2/4 signaling (19
), but conversely, that Atoh1 counters these effects by inhibiting the expression of many differentiation-specific genes, BMP targets among them.
Most striking, the enforced co-expression of Atoh1 and Gli1 in primary GNPs explanted from the early postnatal cerebella of healthy C57BL/6 mice guaranteed their conversion into tumor-initiating cells, less than 200 of which induced MBs within two weeks of transplantation into the brains of naïve recipient animals. Remarkably, Gli1 and Atoh1 together appear sufficient to transform primary GNPs into MB-initiating cells. The fact that Atoh1 alone was incapable of inducing MBs in this assay but maximized the oncogenic potential of Gli1 underscores how anti-differentiative and pro-proliferative programs can synergize in initiating cancer.