Proliferation Of Granule Cell Progenitors Research Articles

An EED/PRC2-H19 Loop Regulates Cerebellar Development.

EED (embryonic ectoderm development) is a core subunit of the polycomb repressive complex 2 (PRC2), which senses the trimethylation of histone H3 lysine 27 (H3K27). However, its biological function in cerebellar development remains unknown. Here, we show that EED deletion from neural stem cells (NSCs) or cerebellar granule cell progenitors (GCPs) leads to reduced GCPs proliferation, cell death, cerebellar hypoplasia, and motor deficits in mice. Joint profiling of transcripts and ChIP-seq analysis in cerebellar granule cells reveals that EED regulates bunches of genes involved in cerebellar development. EED ablation exhibits overactivation of a developmental repressor long non-coding RNA H19. Importantly, an obvious H3K27ac enrichment is found at Ctcf, a trans-activator of H19, and H3K27me3 enrichment at the H19 imprinting control region (ICR), suggesting that EED regulates H19 in an H3K27me3-dependent manner. Intriguingly, H19 deletion reduces EED expression and the reprogramming of EED-mediated H3K27me3 profiles, resulting in increased proliferation, differentiation, and decreased apoptosis of GCPs. Finally, molecular and genetic evidence provides that increased H19 expression is responsible for cerebellar hypoplasia and motor defects in EED mutant mice. Thus, this study demonstrates that EED, H19 forms a negative feedback loop, which plays a crucial role in cerebellar morphogenesis and controls cerebellar development.

MDB-28. THE CYTOSKELETON REGULATORS FORMINS CONTROL THE SHH PATHWAY AND ARE INVOLVED IN MEDULLOBLASTOMA TUMORIGENESIS

Abstract BACKGROUND Medulloblastoma (MB) is a lethal pediatric malignancy of the cerebellum. Among MB’s molecular subgroups, Sonic Hedgehog (SHH) is the most abundant and it is characterized by alterations of key components of the SHH development pathway. However, the molecular mechanisms driving SHH-MB require to further be unveiled to design more effective therapies. Recently, we identified formins, well-known players of cytoskeletal dynamics, as regulators of SHH signaling able to affect SHH-MB growth. METHODS Luciferase functional assays, western-blot and RT-qPCR have been performed to analyze the effect of formins on GLI1 (the final effector of SHH signaling) transcriptional activity and expression. Proliferation assays and Brillouin microscopy have been carried out on primary murine SHH-MB cells to test the effect of formins on SHH-MB growth and stiffness. RESULTS We demonstrated that formins have a dual role in the regulation of SHH signaling, either positive or negative. The modulation of formins expression in SHH-dependent cell models affect GLI1 transcriptional activity and expression. Moreover, formins show opposite protein expression levels during the development of murine cerebellum, and their modulation impair the proliferation of granule cell progenitors (GCPs), the cells of origin of MB. Interestingly, formins protein levels are highly altered in SHH-MB samples. Notably, the overexpression or the genetic silencing of formins in SHH-MB primary cells from Math1-Cre/Ptc1fl/fl mice significantly impact on the cell proliferation as consequence of modulated GLI1 expression levels and the stiffness of primary SHH-MB cells, thus suggesting that formins could affect tumor cell motility and invasiveness. CONCLUSIONS Our findings identify formins as regulators of SHH signaling and illuminate on the role of cytoskeleton in SHH-MB for the design of innovative interventions.

Disruption of protein geranylgeranylation in the cerebellum causes cerebellar hypoplasia and ataxia via blocking granule cell progenitor proliferation

The prenylation of proteins is involved in a variety of biological functions. However, it remains unknown whether it plays an important role in the morphogenesis of the cerebellum. To address this question, we generated a mouse model, in which the geranylgeranyl pyrophosphate synthase (Ggps1) gene is inactivated in neural progenitor cells in the developing cerebellum. We report that conditional knockout (cKO) of Ggps1 leads to severe ataxia and deficient locomotion. To identify the underlying mechanisms, we completed a series of cellular and molecular experiments. First, our morphological analysis revealed significantly decreased population of granule cell progenitors (GCPs) and impaired proliferation of GCPs in the developing cerebellum of Ggps1 cKO mice. Second, our molecular analysis showed increased expression of p21, an important cell cycle regulator in Ggps1 cKO mice. Together, this study highlights a critical role of Ggpps-dependent protein prenylation in the proliferation of cerebellar GCPs during cerebellar development.

Pleiotrophin, nitric oxide and glutamate AMPA receptors in chick cerebellum morphogenesis.

Avian cerebellum, a highly conserved, laminated and foliated structure, provides an excellent model for developmental studies. During the intermediate embryonic stages, granule cell progenitor proliferation and the inwards migration of post-mitotic granule cells have been implicated in the morphogenesis of cerebellar cortex cytoarchitecture and foliation. The present study questioned the spatio-temporal expression pattern of pleiotrophin, an extracellular matrix growth factor, during the morphogenesis of embryonic cerebellum, and the roles of ionotropic AMPA glutamate receptors and the diffusible neuromodulator nitric oxide (NO) in the proliferation pattern of EGL granule cell progenitors. To this end, the density of proliferating cells in the developing embryonic external granule layer (EGL) was determined following acute treatment with AMPA receptor antagonist CNQX or NO synthase inhibitor L-NAME, at embryonic stages HH38-41 (E12-E15 days), by means of BrdU immunohistochemistry and double immunofluorescence. Importantly, at earlier stages, pleiotrophin-like immunoreactivity showed high expression levels in the EGL that gradually decreased, persisting within the growing folia apices, later in development. Interestingly, blockage of AMPA receptors had no effect; while NOS inhibition resulted in transient age- and region-specific increases of EGL granule progenitor cell proliferation at earlier stages, but decreased the post-mitotic granule cells at folia apices, at a later stage HH41 (E15 day). Overall, NO had a transient anti-proliferative effect in EGL similar to mammalian cerebellum, acting as a modulator of the EGL function at different stages, suggesting its possible implication in complex processes guiding cerebellar cytoarchitecture and folia formation.

MBRS-64. STUDY OF ARGININE METHYL TRANSFERASES IN MEDULLOBLASTOMA

Medulloblastoma is the most common malignant pediatric cancer and a leading cause of childhood cancer-related mortality. There is dire need for new therapies. Molecular sub-classification of medulloblastomas has identified chromatin modifiers as potential drivers of tumorigenesis. Expression of the RE1 Silencing Transcription Factor (REST), a hub for assembly of repressive chromatin remodelers and a known regulator of neurogenesis, is elevated in a subset of human sonic hedgehog (SHH) subgroup medulloblastomas, and is associated with poor prognosis. Using a novel transgenic mouse model, we showed REST to be a driver of medulloblastoma development. Surprisingly, our studies also revealed a role for REST in promoting proliferation of granule cell progenitors (GCPs), the cells of origin of SHH-driven medulloblastoma, and a concomitant loss of telomeres and increased genomic instability. We performed a gain of function screen using a library of chromatin modifiers to understand the mechanism by which proliferative potential was maintained, despite the loss of telomeres. This screen identified the Protein Arginine Methyltransferase 6 (PRMT6) as a high confidence hit. PRMT6 upregulation caused a reduction in CDKN2A, an important regulator of replicative senescence. Evasion of senescence is frequently implicated in tumor progression. Using a chemical screen, we also identified a novel, selective, reversible and competitive inhibitor of PRMT6. The consequence of genetic and pharmacological inhibition of PRMT6 on cell proliferation and senescence will be reported. Thus, our studies are the first to demonstrate a role for arginine methyl transferase family of chromatin modifiers in medulloblastoma genesis.

Mosaic Analysis with Double Markers reveals IGF1R function in granule cell progenitors during cerebellar development

During cerebellar development, granule cell progenitors (GCPs) proliferate exponentially for a fixed period, promoted by paracrine mitogenic factor Sonic Hedgehog (Shh) secreted from Purkinje cells (PCs). Dysregulation of Shh signaling leads to uncontrolled GCP proliferation and medulloblastoma. Serendipitously our previous work discovered insulin-like growth factor 1 (IGF1) as another key driver for medulloblastoma, which led to the current investigation into the role of IGF1 in GCPs during normal development. While the IGF1R conditional knockout model revealed GCP defects in anterior cerebellum, the posterior cerebellum was mostly intact, likely owing to incomplete excision of floxed alleles. To circumvent this hurdle, we enlisted a mouse genetic system called Mosaic Analysis of Double Markers (MADM), which sporadically generates homozygous null cells unequivocally labeled with GFP and their wildtype sibling cells labeled with RFP, enabling phenotypic analysis at single-cell resolution. Using MADM, we found that loss of IGF1R resulted in a 10-fold reduction of GCs in both anterior and posterior cerebellum; and that hindered S phase entry and increased cell cycle exit collectively led to this phenotype. Genetic interaction studies showed that IGF1 signaling prevents GCP cell cycle exit at least partially through suppressing the level of p27kip1, a negative regulator of cell cycle. Finally, we found that IGF1 is produced by PCs in a temporally regulated fashion: it is highly expressed early in development when GCPs proliferate exponentially, then gradually decline as GCPs commit to cell cycle exit. Taken together, our studies reveal IGF1 as a paracrine factor that positively regulates GCP cell cycle in cooperation with Shh, through dampening the level of p27 to prevent precocious cell cycle exit. Our work not only showcases the power of phenotypic analysis by the MADM system but also provides an excellent example of multi-factorial regulation of robust developmental programs.

Suppressor of fused controls cerebellum granule cell proliferation by suppressing Fgf8 and spatially regulating Gli proteins.

Cerebellar granule cell (GC) development relies on precise regulation of sonic hedgehog (Shh)-Gli signalling activity, failure of which is associated with motor disorders and medulloblastoma. Mutations in the pathway regulator suppressor of fused (Sufu), which modulates Gli activators and repressors, are linked to cerebellar dysfunction and tumourigenesis. The mechanism by which Sufu calibrates Shh signalling in GCs is unknown. Math1-Cre-mediated deletion of Sufu in mouse GC progenitors (GCPs) demonstrated that Sufu restricts GCP proliferation and promotes cell cycle exit, by promoting expression of Gli3R and suppressing Gli2 levels. Sufu is also required to promote a high threshold of pathway activity in GCPs. Remarkably, central cerebellar lobules are more deleteriously impacted by Sufu deletion, but are less sensitive to downstream genetic manipulations to reduce Gli2 expression or overexpress a Gli3R mimic, compared with anterior lobules. Transcriptome sequencing uncovered new Sufu targets, especially Fgf8, which is upregulated in Sufu-mutant GCPs. We demonstrate that Fgf8 is necessary and sufficient to drive Sufu-mutant GCP proliferation. This study reveals new insights into the spatial and temporal regulation of cerebellar Shh-Gli signalling, while uncovering new targets, such as Fgf8.

GENE-44. BRD4 DELETION LEADS TO CEREBELLAR DEFICITS AND ATAXIA

Abstract Cerebellar neuronal progenitors undergo a series of divisions before irreversibly exiting the cell cycle and differentiating into neurons. Dysfunction of this process underlies many neurological diseases including ataxia and the most common pediatric brain tumor, medulloblastoma. To better define the pathways controlling the most abundant neuronal cells in the mammalian cerebellum, cerebellar granule cell progenitors (GCPs), we performed RNA-sequencing of GCPs exiting the cell cycle. Time-series modeling of GCP cell cycle exit identified downregulation of activity of the epigenetic reader protein Brd4. Brd4 binding to the Gli1 locus is controlled by Casein Kinase 1δ (CK1 δ-dependent phosphorylation during GCP proliferation, and decreases during GCP cell cycle exit. Importantly, conditional deletion of Brd4 in vivo in the developing cerebellum induces cerebellar morphological deficits and ataxia. These studies define an essential role for Brd4 in cerebellar granule cell neurogenesis and are critical for designing clinical trials utilizing Brd4 inhibitors in neurological indications.

Time series modeling of cell cycle exit identifies Brd4 dependent regulation of cerebellar neurogenesis

Cerebellar neuronal progenitors undergo a series of divisions before irreversibly exiting the cell cycle and differentiating into neurons. Dysfunction of this process underlies many neurological diseases including ataxia and the most common pediatric brain tumor, medulloblastoma. To better define the pathways controlling the most abundant neuronal cells in the mammalian cerebellum, cerebellar granule cell progenitors (GCPs), we performed RNA-sequencing of GCPs exiting the cell cycle. Time-series modeling of GCP cell cycle exit identified downregulation of activity of the epigenetic reader protein Brd4. Brd4 binding to the Gli1 locus is controlled by Casein Kinase 1δ (CK1 δ)-dependent phosphorylation during GCP proliferation, and decreases during GCP cell cycle exit. Importantly, conditional deletion of Brd4 in vivo in the developing cerebellum induces cerebellar morphological deficits and ataxia. These studies define an essential role for Brd4 in cerebellar granule cell neurogenesis and are critical for designing clinical trials utilizing Brd4 inhibitors in neurological indications.

Basal Suppression of the Sonic Hedgehog Pathway by the G-Protein-Coupled Receptor Gpr161 Restricts Medulloblastoma Pathogenesis

Sonic hedgehog (Shh) determines cerebellar granule cell (GC) progenitor proliferation and medulloblastoma pathogenesis. However, the pathways regulating GC progenitors during embryogenesis before Shh production by Purkinje neurons and their roles in tumorigenesis remain unclear. The cilium-localized G-protein-coupled receptor Gpr161 suppresses Shh-mediated signaling in the neural tube. Here, bydeleting Gpr161 in mouse neural stem cells orGC progenitors, we establish Gpr161 as a tumor suppressor in Shh subtype medulloblastoma. Irrespective of Shh production in the cerebellum, Gpr161 deletion increased downstream activity of the Shh pathway by restricting Gli3-mediated repression, causing more extensive generation and proliferation of GC progenitors. Moreover, earlier deletion of Gpr161 during embryogenesis increased tumor incidence and severity. GC progenitor overproduction during embryogenesis from Gpr161 deletion was cilium dependent, unlike normal development. Low GPR161 expression correlated with poor survival ofSHH subtype medulloblastoma patients. Gpr161 restricts GC progenitor production by preventing premature and Shh-dependent pathway activity, highlighting the importance of basal pathway suppression in tumorigenesis.

Maml1 acts cooperatively with Gli proteins to regulate sonic hedgehog signaling pathway

Sonic hedgehog (Shh) signaling is essential for proliferation of cerebellar granule cell progenitors (GCPs) and its misregulation is linked to various disorders, including cerebellar cancer medulloblastoma. The effects of Shh pathway are mediated by the Gli family of transcription factors, which controls the expression of a number of target genes, including Gli1. Here, we identify Mastermind-like 1 (Maml1) as a novel regulator of the Shh signaling since it interacts with Gli proteins, working as a potent transcriptional coactivator. Notably, Maml1 silencing results in a significant reduction of Gli target genes expression, with a negative impact on cell growth of NIH3T3 and Patched1−/− mouse embryonic fibroblasts (MEFs), bearing a constitutively active Shh signaling. Remarkably, Shh pathway activity results severely compromised both in MEFs and GCPs deriving from Maml1−/− mice with an impairment of GCPs proliferation and cerebellum development. Therefore Maml1−/− phenotype mimics aspects of Shh pathway deficiency, suggesting an intrinsic requirement for Maml1 in cerebellum development. The present study shows a new role for Maml1 as a component of Shh signaling, which plays a crucial role in both development and tumorigenesis.

Casein kinase 1δ is an APC/C(Cdh1) substrate that regulates cerebellar granule cell neurogenesis.

Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of CNS progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/C(Cdh1)) ubiquitin ligase, and conditional deletion of the APC/C(Cdh1) activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/C(Cdh1) also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/C(Cdh1) controls CK1δ levels to balance proliferation and cell-cycle exit in the developing CNS. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a therapeutic target.

CB-14 * GENOMIC APPROACHES TO IDENTIFY CODING AND NONCODING RNA TARGETS IN MEDULLOBLASTOMA

Medulloblastoma is the most common malignant pediatric brain tumor. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from this disease. Medulloblastoma includes four groups (SHH, WNT, Group 3 and Group 4), which show differences in karyotype, histology, and prognosis. Since some of these tumors originate from granule cell progenitors (GCPs) of the cerebellum a better understanding of the mechanisms controlling progenitor cycle exit and differentiation could unravel new promising targets for medulloblastoma. To gain insight into the mechanisms controlling GCP proliferation and differentiation, we performed RNA sequencing of GCPs at different stages of differentiation. These cells originate in the external germinal layer (EGL) of the cerebellum, undergo predominantly symmetric divisions during early postnatal EGL development, and exit the cell cycle within a narrow time frame. We studied GCP cell cycle exit and obtained several differentially expressed lncRNAs governing neuronal differentiation. We demonstrate that some of these lncRNAs are involved in GCP cell cycle exit and are potentially dysregulated in mouse models of medulloblastoma. Further, we demonstrate that the Anaphase Promoting Complex/cyclosome (APC/C) is also potentially dysregulated in medulloblastoma, thus leading to uncontrolled cell proliferation via lncRNA expression. These studies underscore a previously unappreciated role for APC/C in controlling epigenetic pathways in medulloblastoma.

WNT3 Inhibits Cerebellar Granule Neuron Progenitor Proliferation and Medulloblastoma Formation via MAPK Activation

During normal cerebellar development, the remarkable expansion of granule cell progenitors (GCPs) generates a population of granule neurons that outnumbers the total neuronal population of the cerebral cortex, and provides a model for identifying signaling pathways that may be defective in medulloblastoma. While many studies focus on identifying pathways that promote growth of GCPs, a critical unanswered question concerns the identification of signaling pathways that block mitogenic stimulation and induce early steps in differentiation. Here we identify WNT3 as a novel suppressor of GCP proliferation during cerebellar development and an inhibitor of medulloblastoma growth in mice. WNT3, produced in early postnatal cerebellum, inhibits GCP proliferation by down-regulating pro-proliferative target genes of the mitogen Sonic Hedgehog (SHH) and the bHLH transcription factor Atoh1. WNT3 suppresses GCP growth through a non-canonical Wnt signaling pathway, activating prototypic mitogen-activated protein kinases (MAPKs), the Ras-dependent extracellular-signal-regulated kinases 1/2 (ERK1/2) and ERK5, instead of the classical β-catenin pathway. Inhibition of MAPK activity using a MAPK kinase (MEK) inhibitor reversed the inhibitory effect of WNT3 on GCP proliferation. Importantly, WNT3 inhibits proliferation of medulloblastoma tumor growth in mouse models by a similar mechanism. Thus, the present study suggests a novel role for WNT3 as a regulator of neurogenesis and repressor of neural tumors.

Analysis of human samples reveals impaired SHH-dependent cerebellar development in Joubert syndrome/Meckel syndrome

Joubert syndrome (JS) and Meckel syndrome (MKS) are pleiotropic ciliopathies characterized by severe defects of the cerebellar vermis, ranging from hypoplasia to aplasia. Interestingly, ciliary conditional mutant mice have a hypoplastic cerebellum in which the proliferation of cerebellar granule cell progenitors (GCPs) in response to Sonic hedgehog (SHH) is severely reduced. This suggests that Shh signaling defects could contribute to the vermis hypoplasia observed in the human syndromes. As existing JS/MKS mutant mouse models suggest apparently contradictory hypotheses on JS/MKS etiology, we investigated Shh signaling directly on human fetal samples. First, in an examination of human cerebellar development, we linked the rates of GCP proliferation to the different levels and localizations of active Shh signaling and showed that the GCP possessed a primary cilium with CEP290 at its base. Second, we found that the proliferation of GCPs and their response to SHH were severely impaired in the cerebellum of subjects with JS/MKS and Jeune syndrome. Finally, we showed that the defect in GCP proliferation was similar in the cerebellar vermis and hemispheres in all patients with ciliopathy analyzed, suggesting that the specific cause of vermal hypo-/aplasia precedes this defect. Our results, obtained from the analysis of human samples, show that the hemispheres and the vermis are affected in JS/MKS and provide evidence of a defective cellular mechanism in these pathologic processes.

Impaired motor coordination and disrupted cerebellar architecture in Fgfr1 and Fgfr2 double knockout mice

Fibroblast growth factor receptor (FGFR) signaling determines the size of the cerebral cortex by regulating the amplification of radial glial stem cells, and participates in the formation of midline glial structures. We show that Fgfr1 and Fgfr2 double knockouts (FGFR DKO) generated by Cre-mediated recombination driven by the human GFAP promoter (hGFAP) have reduced cerebellar size due to reduced proliferation of radial glia and other glial precursors in late embryonic and neonatal FGFR DKO mice. The proliferation of granule cell progenitors (GCPs) in the EGL was also reduced, leading to reduced granule cell numbers. Furthermore, both inward migration of granule cells into the inner granule cell layer (IGL) and outward migration of GABA interneurons into the molecular layer (ML) were arrested, disrupting layer and lobular morphology. Purkinje neurons and their dendrites, which were not targeted by Cre-mediated recombination of Fgf receptors, were also misplaced in FGFR DKO mice, possibly as a consequence of altered Bergmann glia orientation or reduced granule cell number. Our findings indicate a dual role for FGFR signaling in cerebellar morphogenesis. The first role is to amplify the number of granule neuron precursors in the external granular layer and glial precursor cells throughout the cerebellum. The second is to establish the correct Bergmann glia morphology, which is crucial for granule cell migration. The disrupted cerebellar size and laminar architecture resulting from loss of FGFR signaling impair motor learning and coordination in FGFR DKO mice.

Bmi1 overexpression in the cerebellar granule cell lineage of mice affects cell proliferation and survival without initiating medulloblastoma formation.

SUMMARYBMI1 is a potent inducer of neural stem cell self-renewal and neural progenitor cell proliferation during development and in adult tissue homeostasis. It is overexpressed in numerous human cancers – including medulloblastomas, in which its functional role is unclear. We generated transgenic mouse lines with targeted overexpression of Bmi1 in the cerebellar granule cell lineage, a cell type that has been shown to act as a cell of origin for medulloblastomas. Overexpression of Bmi1 in granule cell progenitors (GCPs) led to a decrease in cerebellar size due to decreased GCP proliferation and repression of the expression of cyclin genes, whereas Bmi1 overexpression in postmitotic granule cells improved cell survival in response to stress by altering the expression of genes in the mitochondrial cell death pathway and of Myc and Lef-1. Although no medulloblastomas developed in ageing cohorts of transgenic mice, crosses with Trp53−/− mice resulted in a low incidence of medulloblastoma formation. Furthermore, analysis of a large collection of primary human medulloblastomas revealed that tumours with a BMI1high TP53low molecular profile are significantly enriched in Group 4 human medulloblastomas. Our data suggest that different levels and timing of Bmi1 overexpression yield distinct cellular outcomes within the same cellular lineage. Importantly, Bmi1 overexpression at the GCP stage does not induce tumour formation, suggesting that BMI1 overexpression in GCP-derived human medulloblastomas probably occurs during later stages of oncogenesis and might serve to enhance tumour cell survival.

Sequential Activation of Snail1 and N-Myc Modulates Sonic Hedgehog–Induced Transformation of Neural Cells

Activation of the Sonic hedgehog (Shh) pathway and increased expression of Gli1 play an important role in proliferation and transformation of granule cell progenitors (GCP) in the developing cerebellum. Medulloblastomas arising from cerebellar GCPs are frequently driven by Shh pathway-activating mutations; however, molecular mechanisms of Shh pathway dysregulation and transformation of neural progenitors remain poorly defined. We report that the transcription factor and oncogene Snail1 (Sna1) is directly induced by Shh pathway activity in GCPs, murine medulloblastomas, and human medulloblastoma cells. Enforced expression of Sna1 was sufficient to induce GCPs and medulloblastoma cell proliferation in the absence of Shh/Gli1 exposure. In addition, enforced expression of Sna1 increased transformation of medulloblastoma cells in vitro and in vivo. Analysis of potential Sna1 targets in neural cells revealed a novel Sna1 target, N-Myc, a transcription factor known to play a role in Shh-mediated GCP proliferation and medulloblastoma formation. We found that Sna1 directly induced transcription of N-Myc in human medulloblastoma cells and that depletion of N-Myc ablated the Sna1-induced proliferation and transformation. Taken together, these results provide further insight into the mechanism of Shh-induced transformation of neural progenitor cells and suggest that induction of Sna1 may serve to amplify the oncogenic potential of Shh pathway activation through N-Myc induction.

Multiple developmental programs are altered by loss ofZic1andZic4to cause Dandy-Walker malformation cerebellar pathogenesis

Heterozygous deletions encompassing the ZIC1;ZIC4 locus have been identified in a subset of individuals with the common cerebellar birth defect Dandy-Walker malformation (DWM). Deletion of Zic1 and Zic4 in mice produces both cerebellar size and foliation defects similar to human DWM, confirming a requirement for these genes in cerebellar development and providing a model to delineate the developmental basis of this clinically important congenital malformation. Here, we show that reduced cerebellar size in Zic1 and Zic4 mutants results from decreased postnatal granule cell progenitor proliferation. Through genetic and molecular analyses, we show that Zic1 and Zic4 have Shh-dependent function promoting proliferation of granule cell progenitors. Expression of the Shh-downstream genes Ptch1, Gli1 and Mycn was downregulated in Zic1/4 mutants, although Shh production and Purkinje cell gene expression were normal. Reduction of Shh dose on the Zic1(+/-);Zic4(+/-) background also resulted in cerebellar size reductions and gene expression changes comparable with those observed in Zic1(-/-);Zic4(-/-) mice. Zic1 and Zic4 are additionally required to pattern anterior vermis foliation. Zic mutant folial patterning abnormalities correlated with disrupted cerebellar anlage gene expression and Purkinje cell topography during late embryonic stages; however, this phenotype was Shh independent. In Zic1(+/-);Zic4(+/-);Shh(+/-), we observed normal cerebellar anlage patterning and foliation. Furthermore, cerebellar patterning was normal in both Gli2-cko and Smo-cko mutant mice, where all Shh function was removed from the developing cerebellum. Thus, our data demonstrate that Zic1 and Zic4 have both Shh-dependent and -independent roles during cerebellar development and that multiple developmental disruptions underlie Zic1/4-related DWM.

21-P044 Fate specification of glial progenitors but not proliferation of granule cell progenitors is controlled by Bmi1 through BMP pathway inhibition during cerebellar development

21-P044 Fate specification of glial progenitors but not proliferation of granule cell progenitors is controlled by Bmi1 through BMP pathway inhibition during cerebellar development