TMOD-26. MYC OVEREXPRESSION AND SMARCA4 LOSS IN GRANULE CELL PRECURSORS COOPERATE TO DRIVE MEDULLOBLASTOMA FORMATION IN MICE

Mutations in SMARCA4 are frequently identified in medulloblastoma, the most common pediatric malignant brain tumor. However, the functional significance of these mutations and their suitability as a therapeutic target remain largely unclear. Medulloblastomas are divided into 4 subgroups according to their localization, molecular biology, and clinical course: WNT, SHH, Group 3, and Group 4. Group 3 medulloblastomas are associated with the poorest outcome and frequently show amplifications of the oncogene MYC. Additionally, SMARCA4 is mutated in around 15 % of cases. The few mouse models developed for this entity so far all involve the overexpression of MYC, mostly in combination with other drivers. However, none of these models include alterations in Smarca4. In our approach, we combined an overexpression of MYC with a loss of SMARCA4 in granule cell precursors, which successfully induced tumor formation in mice. For this purpose, granule cell precursors were isolated from 7-day-old Math1-creER T2 ::Smarca4 fl/fl pups after tamoxifen induced loss of SMARCA4. MYC overexpression was achieved by lentiviral transduction and transduced cells were transplanted into immunodeficient CD1 nu/nu mice. Preliminary results within a small cohort showed tumor formation in 5/19 transplanted mice (26 %) after 6 months. Immunohistochemically, tumors all stained negative for SMARCA4. In a next step, additional cohorts will elucidate if tumor development is indeed accelerated by or even dependent on the loss of SMARCA4. Additionally, the neoplastic potential of tumor cells will be verified with the aid of secondary recipient mice. To evaluate to what extent the generated tumors are comparable to human Group 3 medulloblastomas, tumors will be extensively analyzed on a morphological, transcriptional, and epigenetic level. Altogether, we hope to establish a suitable mouse model for SMARCA4 mutated Group 3 medulloblastoma that will help to elucidate the role of SMARCA4 in tumor development and to identify new therapeutic targets.

CHARGE syndrome protein CHD7 regulates epigenomic activation of enhancers in granule cell precursors and gyrification of the cerebellum

Regulation of chromatin plays fundamental roles in the development of the brain. Haploinsufficiency of the chromatin remodeling enzyme CHD7 causes CHARGE syndrome, a genetic disorder that affects the development of the cerebellum. However, how CHD7 controls chromatin states in the cerebellum remains incompletely understood. Using conditional knockout of CHD7 in granule cell precursors in the mouse cerebellum, we find that CHD7 robustly promotes chromatin accessibility, active histone modifications, and RNA polymerase recruitment at enhancers. In vivo profiling of genome architecture reveals that CHD7 concordantly regulates epigenomic modifications associated with enhancer activation and gene expression of topologically-interacting genes. Genome and gene ontology studies show that CHD7-regulated enhancers are associated with genes that control brain tissue morphogenesis. Accordingly, conditional knockout of CHD7 triggers a striking phenotype of cerebellar polymicrogyria, which we have also found in a case of CHARGE syndrome. Finally, we uncover a CHD7-dependent switch in the preferred orientation of granule cell precursor division in the developing cerebellum, providing a potential cellular basis for the cerebellar polymicrogyria phenotype upon loss of CHD7. Collectively, our findings define epigenomic regulation by CHD7 in granule cell precursors and identify abnormal cerebellar patterning upon CHD7 depletion, with potential implications for our understanding of CHARGE syndrome.

The Eya1 Phosphatase Mediates Shh-Driven Symmetric Cell Division of Cerebellar Granule Cell Precursors

During neural development, stem and precursor cells can divide either symmetrically or asymmetrically. The transition between symmetric and asymmetric cell divisions is a major determinant of precursor cell expansion and neural differentiation, but the underlying mechanisms that regulate this transition are not well understood. Here, we identify the Sonic hedgehog (Shh) pathway as a critical determinant regulating the mode of division of cerebellar granule cell precursors (GCPs). Using partial gain and loss of function mutations within the Shh pathway, we show that pathway activation determines spindle orientation of GCPs, and that mitotic spindle orientation correlates with the mode of division. Mechanistically, we show that the phosphatase Eya1 is essential for implementing Shh-dependent GCP spindle orientation. We identify atypical protein kinase C (aPKC) as a direct target of Eya1 activity and show that Eya1 dephosphorylates a critical threonine (T410) in the activation loop. Thus, Eya1 inactivates aPKC, resulting in reduced phosphorylation of Numb and other components that regulate the mode of division. This Eya1-dependent cascade is critical in linking spindle orientation, cell cycle exit and terminal differentiation. Together these findings demonstrate that a Shh-Eya1 regulatory axis selectively promotes symmetric cell divisions during cerebellar development by coordinating spindle orientation and cell fate determinants.

Intrinsic neuronal activity during migration controls the recruitment of specific interneuron subtypes in the postnatal mouse olfactory bulb

Neuronal activity has been identified as a key regulator of neuronal network development, but the impact of activity on migration and terminal positioning of interneuron subtypes is poorly understood. The absence of early subpopulation markers and the presence of intermingled migratory and post-migratory neurons makes the developing cerebral cortex a difficult model to answer these questions. Postnatal neurogenesis in the subventricular zone offers a more accessible and compartmentalized model. Neural stem cells regionalized along the border of the lateral ventricle produce two main subtypes of neural progenitors, granule cells and periglomerular neurons that migrate tangentially in the rostral migratory stream before migrating radially in the OB layers. Here we take advantage of targeted postnatal electroporation to compare the migration of these two population. We do not observe any obvious differences regarding the mode of tangential or radial migration between these two subtypes. However, we find a very striking increase of intrinsic calcium activity only in granule cell precursors when they switch from tangential to radial migration. By decreasing neuronal excitability in granule cell precursors, we find that neuronal activity is critical for normal migratory speed at the end of tangential migration. Importantly, we also find that activity is required for normal positioning and survival of granule cell precursors in the OB layers. Strikingly, decreasing activity of periglomerular neuron precursors did not impact their positioning or survival. Altogether these findings suggest that neuronal excitability plays a subtype specific role during the late stage of migration of postnatally born olfactory bulb interneurons.Significance StatementWhile neuronal activity is a critical factor regulating different aspects of neurogenesis, it has been challenging to study its role during the migration of different neuronal subpopulations. Here, we use postnatal targeted electroporation to label and manipulate the two main olfactory bulb interneuron subpopulations during their migration: granule cell and periglomerular neuron precursors. We find a very striking increase of calcium activity only in granule cell precursors when they switch from tangential to radial migration. Interestingly, blocking activity in granule cell precursors affected their migration, positioning and survival while periglomerular neuron precursors are not affected. These results suggest that neuronal activity is required specifically for the recruitment of granule cell precursors in the olfactory bulb layers.

SMO-M2 mutation does not support cell-autonomous Hedgehog activity in cerebellar granule cell precursors

Growth and patterning of the cerebellum is compromised if granule cell precursors do not properly expand and migrate. During embryonic and postnatal cerebellar development, the Hedgehog pathway tightly regulates granule cell progenitors to coordinate appropriate foliation and lobule formation. Indeed, granule cells impairment or defects in the Hedgehog signaling are associated with developmental, neurodegenerative and neoplastic disorders. So far, scant and inefficient cellular models have been available to study granule cell progenitors, in vitro. Here, we validated a new culture method to grow postnatal granule cell progenitors as hedgehog-dependent neurospheres with prolonged self-renewal and ability to differentiate into granule cells, under appropriate conditions. Taking advantage of this cellular model, we provide evidence that Ptch1-KO, but not the SMO-M2 mutation, supports constitutive and cell-autonomous activity of the hedgehog pathway.

Mitotic granule cell precursors undergo highly dynamic morphological transitions throughout the external germinal layer of the chick cerebellum

The developing cerebellum of amniotes is characterised by a unique, transient, secondary proliferation zone: the external germinal layer (EGL). The EGL is comprised solely of granule cell precursors, whose progeny migrate inwardly to form the internal granule cell layer. While a range of cell morphologies in the EGL has long been known, how they reflect the cells’ differentiation status has previously only been inferred. Observations have suggested a deterministic maturation from outer to inner EGL that we wished to test experimentally. To do this, we electroporated granule cell precursors in chick with plasmids encoding fluorescent proteins and probed labelled cells with markers of both proliferation (phosphohistone H3) and differentiation (Axonin1/TAG1 and NeuroD1). We show that granule cell precursors can display a range of complex forms throughout the EGL while mitotically active. Overexpression of full length NeuroD1 within granule cell precursors does not abolish proliferation, but biases granule cells towards precocious differentiation, alters their migration path and results in a smaller and less foliated cerebellum. Our results show that granule cells show a greater flexibility in differentiation than previously assumed. We speculate that this allows the EGL to regulate its proliferative activity in response to overall patterns of cerebellar growth.