The cerebral cortex develops through the coordinated generation of dozens of neuronal subtypes, but the mechanisms involved remain unclear. Here we show that mouse embryonic stem cells, cultured without any morphogen but in the presence of a sonic hedgehog inhibitor, recapitulate in vitro the major milestones of cortical development, leading to the sequential generation of a diverse repertoire of neurons that display most salient features of genuine cortical pyramidal neurons. When grafted into the cerebral cortex, these neurons develop patterns of axonal projections corresponding to a wide range of cortical layers, but also to highly specific cortical areas, in particular visual and limbic areas, thereby demonstrating that the identity of a cortical area can be specified without any influence from the brain. The discovery of intrinsic corticogenesis sheds new light on the mechanisms of neuronal specification, and opens new avenues for the modelling and treatment of brain diseases.
The classification and pathological mechanisms of many central nervous system inflammatory diseases remain uncertain. In this article we report eight patients with a clinically and radiologically distinct pontine-predominant encephalomyelitis we have named 'chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids' (CLIPPERS). The patients were assessed clinically, radiologically and pathologically at Mayo Clinic, USA and Ghent University Hospital, Belgium from 1999 to 2009. Median follow-up duration from clinical onset was 22 months (range 7-144 months). Patients underwent extensive laboratory (serum and cerebrospinal fluid), radiological and pathological testing (conjunctival, transbronchial and brain biopsies) to search for causes of an inflammatory central nervous system disorder. All eight patients (five female, three male) presented with episodic diplopia or facial paresthesias with subsequent brainstem and occasionally myelopathic symptoms and had a favourable initial response to high dose glucocorticosteroids. All patients had symmetric curvilinear gadolinium enhancement peppering the pons and extending variably into the medulla, brachium pontis, cerebellum, midbrain and occasionally spinal cord. Radiological improvement accompanied clinical response to glucocorticosteroids. Patients routinely worsened following glucocorticosteroid taper and required chronic glucocorticosteroid or other immunosuppressive therapy. Neuropathology of biopsy material from four patients demonstrated white matter perivascular, predominantly T lymphocytic, infiltrate without granulomas, infection, lymphoma or vasculitis. Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids is a definable, chronic inflammatory central nervous system disorder amenable to immunosuppressive treatment. The T cell predominant inflammatory pathology in affected central nervous system lesions and the clinical and radiological response to immunosuppressive therapies is consistent with an immune-mediated process.
During neurogenesis, neural stem/progenitor cells (NPCs) undergo an irreversible fate transition to become neurons. The Notch pathway is important for this process, and repression of Notch-dependent Hes genes is essential for triggering differentiation. However, Notch signaling often remains active throughout neuronal differentiation, implying a change in the transcriptional responsiveness to Notch during the neurogenic transition. We identified Bcl6, an oncogene, as encoding a proneurogenic factor that is required for proper neurogenesis of the mouse cerebral cortex. BCL6 promoted the neurogenic conversion by switching the composition of Notch-dependent transcriptional complexes at the Hes5 promoter. BCL6 triggered exclusion of the co-activator Mastermind-like 1 and recruitment of the NAD(+)-dependent deacetylase Sirt1, which was required for BCL6-dependent neurogenesis. The resulting epigenetic silencing of Hes5 led to neuronal differentiation despite active Notch signaling. Our findings suggest a role for BCL6 in neurogenesis and uncover Notch-BCL6-Sirt1 interactions that may affect other aspects of physiology and disease.
MH designed and performed experiments and interpreted results.. L.A. designed and performed the in vitro experiments, M.A.M., designed and performed the in vivo experiments, L.C-E., the hydroxymethylation and EdU stainings, G.B, experiments with small molecule inhibitors. G.V. and E.A.M. prepared and analysed WGBS and RRHP libraries, analysed RNAseq and interpreted corresponding bionformatic analyses related. N.A., A.R. and S.J.F. performed experiments with β1 integrin model and interpreted results of the p21 models. J.v.d.A. and A.H.B. performed DamID-seq experiments. B.F-C helped on the in vivo analysis. R.A.C. helped on bioinformatics analyses. R.L.M. provided the R26Fucci2a line. F.A. and M.Z.G. performed the live imaging of ductal cells.
Embryonic stem cells (ESCs) constitute a tool of great potential in neurobiology, enabling the directed differentiation of specific neural cell types. We have shown recently that neurons of the cerebral cortex can be generated from mouse ESCs cultured in a chemically defined medium that contains no morphogen, but in the presence of the sonic hedgehog inhibitor cyclopamine. Corticogenesis from ESCs recapitulates the most important steps of cortical development, leading to the generation of multipotent cortical progenitors that sequentially produce cortical pyramidal neurons displaying distinct layer-specific identities. The protocol provides a most reductionist cellular model to tackle the complex mechanisms of cortical development and function, thereby opening new perspectives for the modeling of cortical diseases and the design of novel neurological treatments, while offering an alternative to animal use. In this protocol, we describe a method by which millions of cortical neurons can be generated in 2-3 weeks, starting from a single frozen vial of ESCs.
The development of the cerebral cortex requires the tightly coordinated generation of dozens of neuronal subtypes that will populate specific layers and areas. Recent studies have revealed how pluripotent stem cells (PSC), whether of mouse or human origin, can differentiate into a wide range of cortical neurons in vitro, which can integrate appropriately into the brain following in vivo transplantation. These models are largely artificial but recapitulate a substantial fraction of the complex temporal and regional patterning events that occur during in vivo corticogenesis. Here, we review these findings with emphasis on the new perspectives that they have brought for understanding of cortical development, evolution, and diseases.
Disrupted differentiation during development can lead to oncogenesis, but the underlying mechanisms remain poorly understood. Here we identify BCL6, a transcriptional repressor and lymphoma oncoprotein, as a pivotal factor required for neurogenesis and tumor suppression of medulloblastoma (MB). BCL6 is necessary for and capable of preventing the development of GNP-derived MB in mice, and can block the growth of human MB cells in vitro. BCL6 neurogenic and oncosuppressor effects rely on direct transcriptional repression of Gli1 and Gli2 effectors of the SHH pathway, through recruitment of BCOR corepressor and SIRT1 deacetylase. Our findings identify the BCL6/BCOR/SIRT1 complex as a potent repressor of the SHH pathway in normal and oncogenic neural development, with direct diagnostic and/or therapeutic relevance for SHH MB.
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