Timothy N. Phoenix

The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells

In the developing cerebral cortex, neurons are born on a predictable schedule. Here we show in mice that the essential timing mechanism is programmed within individual progenitor cells, and its expression depends solely on cell-intrinsic and environmental factors generated within the clonal lineage. Multipotent progenitor cells undergo repeated asymmetric divisions, sequentially generating neurons in their normal in vivo order: first preplate cells, including Cajal-Retzius neurons, then deep and finally superficial cortical plate neurons. As each cortical layer arises, stem cells and neuroblasts become restricted from generating earlier-born neuron types. Growth as neurospheres or in co-culture with younger cells did not restore their plasticity. Using short-hairpin RNA (shRNA) to reduce Foxg1 expression reset the timing of mid- but not late-gestation progenitors, allowing them to remake preplate neurons and then cortical-plate neurons. Our data demonstrate that neural stem cells change neuropotency during development and have a window of plasticity when restrictions can be reversed.

Novel mutations target distinct subgroups of medulloblastoma

Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.

C11orf95 – RELA fusions drive oncogenic NF-κB signalling in ependymoma

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95–RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95–RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells—the cell of origin of ependymoma—to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95–RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.

Bmi-1 cooperates with Foxg1 to maintain neural stem cell self-renewal in the forebrain

Neural stem cells (NSCs) persist throughout life in two forebrain areas: the subventricular zone (SVZ) and the hippocampus. Why forebrain NSCs self-renew more extensively than those from other regions remains unclear. Prior studies have shown that the polycomb factor Bmi-1 is necessary for NSC self-renewal and that it represses the cell cycle inhibitors p16, p19, and p21. Here we show that overexpression of Bmi-1 enhances self-renewal of forebrain NSCs significantly more than those derived from spinal cord, demonstrating a regional difference in responsiveness. We show that forebrain NSCs require the forebrain-specific transcription factor Foxg1 for Bmi-1-dependent self-renewal, and that repression of p21 is a focus of this interaction. Bmi-1 enhancement of NSC self-renewal is significantly greater with increasing age and passage. Importantly, when Bmi-1 is overexpressed in cultured adult forebrain NSCs, they expand dramatically and continue to make neurons even after multiple passages, when control NSCs have become restricted to glial differentiation. Together these findings demonstrate the importance of Bmi-1 and Foxg1 cooperation to maintenance of NSC multipotency and self-renewal, and establish a useful method for generating abundant forebrain neurons ex vivo, outside the neurogenic niche.

Medulloblastoma Genotype Dictates Blood Brain Barrier Phenotype.

The childhood brain tumor, medulloblastoma, includes four subtypes with very different prognoses. Here, we show that paracrine signals driven by mutant β-catenin in WNT-medulloblastoma, an essentially curable form of the disease, induce an aberrant fenestrated vasculature that permits the accumulation of high levels of intra-tumoral chemotherapy and a robust therapeutic response. In contrast, SHH-medulloblastoma, a less curable disease subtype, contains an intact blood brain barrier, rendering this tumor impermeable and resistant to chemotherapy. The medulloblastoma-endothelial cell paracrine axis can be manipulated in vivo, altering chemotherapy permeability and clinical response. Thus, medulloblastoma genotype dictates tumor vessel phenotype, explaining in part the disparate prognoses among medulloblastoma subtypes and suggesting an approach to enhance the chemoresponsiveness of other brain tumors.

Spred1, a negative regulator of Ras–MAPK–ERK, is enriched in CNS germinal zones, dampens NSC proliferation, and maintains ventricular zone structure

Neural stem cells (NSCs) have great potential for self-renewal, which must be tightly regulated to generate appropriate cell numbers during development and to prevent tumor formation. The Ras-MAPK-ERK pathway affects mitogen-stimulated proliferation, and negative regulators are likely to be important for keeping self-renewal in check. Sprouty-related protein with an EVH1 domain (Spred1) is a recently discovered negative Ras-MAPK-ERK regulator linked to a neurofibromatosis 1 (NF-1)-like human syndrome; however, its role in CNS development has not been explored. We show that Spred1 is highly enriched in CNS germinal zones during neurogenesis. Spred1 knockdown increases NSC self-renewal and progenitor proliferation cell-autonomously, and overexpression causes premature differentiation. Surprisingly, Spred1 knockdown in vivo in the embryonic mouse forebrain frequently resulted in periventricular heterotopia, developmental abnormalities often associated with mutations in genes in the vesicular trafficking pathway that cause disruption of germinal zones and impair cell migration. In cortical progenitor cells, Spred1 localizes within distinct vesicles, indicating a potential role in transport. Spred1 knockdown gradually leads to disruption of the apical ventricular zone and loss of radial glia alignment. This impairs late neuronal migration, resulting in the formation of periventricular masses. Thus, Spred1 is critical for normal cortical development, as it modulates progenitor self-renewal/proliferation and helps maintain the integrity and organization of germinal zones.

Molecular Characterisation of Transport Mechanisms at the Developing Mouse Blood–CSF Interface: A Transcriptome Approach

Exchange mechanisms across the blood–cerebrospinal fluid (CSF) barrier in the choroid plexuses within the cerebral ventricles control access of molecules to the central nervous system, especially in early development when the brain is poorly vascularised. However, little is known about their molecular or developmental characteristics. We examined the transcriptome of lateral ventricular choroid plexus in embryonic day 15 (E15) and adult mice. Numerous genes identified in the adult were expressed at similar levels at E15, indicating substantial plexus maturity early in development. Some genes coding for key functions (intercellular/tight junctions, influx/efflux transporters) changed expression during development and their expression patterns are discussed in the context of available physiological/permeability results in the developing brain. Three genes: Secreted protein acidic and rich in cysteine (Sparc), Glycophorin A (Gypa) and C (Gypc), were identified as those whose gene products are candidates to target plasma proteins to choroid plexus cells. These were investigated using quantitative- and single-cell-PCR on plexus epithelial cells that were albumin- or total plasma protein-immunopositive. Results showed a significant degree of concordance between plasma protein/albumin immunoreactivity and expression of the putative transporters. Immunohistochemistry identified SPARC and GYPA in choroid plexus epithelial cells in the embryo with a subcellular distribution that was consistent with transport of albumin from blood to cerebrospinal fluid. In adult plexus this pattern of immunostaining was absent. We propose a model of the cellular mechanism in which SPARC and GYPA, together with identified vesicle-associated membrane proteins (VAMPs) may act as receptors/transporters in developmentally regulated transfer of plasma proteins at the blood–CSF interface.

Orthotopic models of pediatric brain tumors in zebrafish

High-throughput screens (HTS) of compound toxicity against cancer cells can identify thousands of potential new drug-leads. But only limited numbers of these compounds can progress to expensive and labor-intensive efficacy studies in mice, creating a ‘bottle neck’ in the drug development pipeline. Approaches that triage drug-leads for further study are greatly needed. Here we provide an intermediary platform between HTS and mice by adapting mouse models of pediatric brain tumors to grow as orthotopic xenografts in the brains of zebrafish. Freshly isolated mouse ependymoma, glioma and choroid plexus carcinoma cells expressing red fluorescence protein were conditioned to grow at 34 °C. Conditioned tumor cells were then transplanted orthotopically into the brains of zebrafish acclimatized to ambient temperatures of 34 °C. Live in vivo fluorescence imaging identified robust, quantifiable and reproducible brain tumor growth as well as spinal metastasis in zebrafish. All tumor xenografts in zebrafish retained the histological characteristics of the corresponding parent mouse tumor and efficiently recruited fish endothelial cells to form a tumor vasculature. Finally, by treating zebrafish harboring ERBB2-driven gliomas with an appropriate cytotoxic chemotherapy (5-fluorouracil) or tyrosine kinase inhibitor (erlotinib), we show that these models can effectively assess drug efficacy. Our data demonstrate, for the first time, that mouse brain tumors can grow orthotopically in fish and serve as a platform to study drug efficacy. As large cohorts of brain tumor-bearing zebrafish can be generated rapidly and inexpensively, these models may serve as a powerful tool to triage drug-leads from HTS for formal efficacy testing in mice.

SPARC/osteonectin, an endogenous mechanism for targeting albumin to the blood-cerebrospinal fluid interface during brain development.

Specialized populations of choroid plexus epithelial cells have previously been shown to be responsible for the transfer of individual plasma proteins from blood to the cerebrospinal fluid (CSF), contributing to their characteristically high concentrations in CSF of the developing brain. The mechanism of this protein transfer remains elusive. Using a marsupial, Monodelphis domestica, we demonstrate that the albumin‐binding protein SPARC (osteonectin/BM‐40/culture‐shock protein) is present in a subset of choroid plexus epithelial cells from its first appearance, throughout development, and into adulthood. The synthesis of SPARC by the lateral ventricular plexus was confirmed with real‐time PCR. The expression level of SPARC was higher in plexuses of younger than older animals. Western blot analysis of the gene product confirmed the quantitative PCR results. The co‐localization of SPARC and albumin shown by immunocytochemistry and its cellular location indicate that this glycoprotein may act as a recognition site for albumin. In addition, the numbers of SPARC‐immunopositive cells and its expression were responsive to experimental changes of albumin concentration in the blood. It is suggested that SPARC may be one of the molecules that govern the uptake and delivery of proteins from blood to the CSF. The results also confirm that protein transfer across the blood–CSF barrier is developmentally and physiologically regulated.

Novel MYC-driven medulloblastoma models from multiple embryonic cerebellar cells

Group3 medulloblastoma (MBG3) that predominantly occur in young children are usually associated with MYC amplification and/or overexpression, frequent metastasis and a dismal prognosis. Physiologically relevant MBG3 models are currently lacking, making inferences related to their cellular origin thus far limited. Using in utero electroporation, we here report that MBG3 mouse models can be developed in situ from different multipotent embryonic cerebellar progenitor cells via conditional expression of Myc and loss of Trp53 function in several Cre driver mouse lines. The Blbp-Cre driver that targets embryonic neural progenitors induced tumors exhibiting a large-cell/anaplastic histopathology adjacent to the fourth ventricle, recapitulating human MBG3. Enforced co-expression of luciferase together with Myc and a dominant-negative form of Trp53 revealed that GABAergic neuronal progenitors as well as cerebellar granule cells give rise to MBG3 with their distinct growth kinetics. Cross-species gene expression analysis revealed that these novel MBG3 models shared molecular characteristics with human MBG3, irrespective of their cellular origin. We here developed MBG3 mouse models in their physiological environment and we show that oncogenic insults drive this MB subgroup in different cerebellar lineages rather than in a specific cell of origin.