Molecular heterogeneity of the ganglionic eminence and its relationship to the birth of neocortical interneurons

Neocortical function and malfunction depends critically on a spectrum of inhibitory interneurons. To gain novel insight in normal brain function and mechanisms leading to diseases, such as epilepsy or schizophrenia, the knowledge about the specific functions of each interneuron type appears crucial. To date, the mystery about the diversity of neocortical interneurons remains still unresolved, even though much effort was devoted to the characterization of mature interneurons in the neocortex. Ten years ago, the discovery of the ganglionic eminence (GE) as origin of neocortical interneurons allowed a novel approach to assess their various identities, and thus prompted researchers to follow the fate of cells accommodated in this region. Despite the knowledge of the source location and its subdivision into three GEs, their molecular identity remained unknown apart from few genes, and consequently unabled the specific isolation of precursor cells. To this purpose, we performed extensive microarray analysis of the three GEs at various developmental timepoints to reveal their molecular heterogeneity and identify novel precursor cell surface markers that would enable their prospective isolation by flow cytometry and subsequent fate assessment in vitro and in vivo. Surprisingly, molecular heterogeneity of the three GEs was restricted to few differentially expressed genes. However, the differences in gene expression became more apparent at late developmental stage that may reflect the increased specification during development. Among the three GEs, the LGE and CGE resembled molecularly each other most, while the MGE and CGE were most distantly related to each other. Furthermore, our genome-wide expression study suggested that the CGE, a structure of so far controversial nature, was rather a unique structure than a fusion of the MGE and LGE. Analysis across developmental timepoints revealed that the MGE underwent tremendous changes in gene expression when compared to the differential expression existing between the homochronic MGE and cerebral cortex. In our microarray screen, we could identify Boc as a novel marker of LGE/CGE-resident precursor cells. Taking advantage of Boc that encodes a cell surface molecule, we could isolate the Boc+ cells from the GE by flow cytometry, and demonstrate that in contrast to Boc– cells, they self-renewed and differentiated into oligodendrocytes, astrocytes, and neurons, and thus identify them as progenitor cells. Furthermore, immunohistochemical studies suggested that Boc+ GE cells may correspond to radial glia, the embryonic neural stem cells. The specific expression of Boc in the LGE and CGE, but not in the MGE during their entire lifetime suggested that Boc is a reliable marker for LGE/CGE-resident precursor cells. To assess the fate of Boc+ GE cells, we performed homotypic transplantations of Boc+ and Boc– cells onto organotypic slice cultures, and observed that in contrast to Boc– cells, Boc+ cells did not migrate from the GE to the neocortex suggesting that Boc+ precursor cells of the GE do not generate neocortical interneurons. In future, additional fate-mapping studies of Boc+ GE cells, such as in vivo transplantation, should shed more light on the ability of Boc+ LGE/CGE precursor cells to generate neocortical interneurons, and dependent on the outcome serve to construct a cell-lineage tree of neocortical interneurons. Thus, Boc will remain a useful tool to investigate developmental processes underlying the generation of neocortical and/or other neurons, such as striatal neurons.

Conversion of mouse neural stem cells into glioma cancer stem cells

Caroline Turk

Gliomas are the most common and lethal primary adult human brain cancers. The most lethal form of glioma is glioblastoma multiforme (GBM). Multiple apparently differentiated cell types are present within these tumours, including varying proportions of astrocytes and oligodendrocytes. The identification of new GBM subtypes, the role of the cell-of-origin and the role of commonly disrupted genes are active areas of investigation. Of particular interest to this laboratory is the recent realisation that GBMs may be driven by and initiated from tumorigenic stem cells. Our goal was to develop a mouse model of the disease based on genetic manipulation of in vitro neural stem (NS) cell lines. Importantly, this would enable us to define the contribution of each oncogenic pathway in altering the self-renewal of these cells. The original hypothesis was that the simultaneous activation of three critical pathways (RTK/PI3K, Rb and p53) would be required to confer a glioma-like phenotype. However, surprisingly we found that forced expression of the commonly amplified cyclin dependent kinase-4 (CDK4) was sufficient to elicit reduced dependency on growth factor signalling and a decreased sensitivity to the differentiation factor BMP4. Moreover, preliminary results indicate that the mRNA expression of the proto-oncogene Akt2 increases in CDK4 transfectants, suggestive of a feedback mechanism. We tested whether constitutive expression of Akt2 together with CDK4 increases the severity of the phenotype and initial results suggest this is the case. These findings provide encouragement that minimal genetic events will be required in order to transform NS cells in vitro. These cells can now be tested for in vivo tumorigenicity following transplantation. Finally, we have also generated CDK4 overexpressing cell lines in a specific NS cell variant which I identified as expressing an oligodendrocyte differentiation bias (MG1-7). We are therefore now in a position to assess whether differentiation behaviour is affected by the disruption of this pathway. Together these findings highlight the utility of mouse NS cells as a tool to study glioma

2009

Expression of FGF-2 in neural progenitor cells enhances their potential for cellular brain repair in the rodent cortex

Michiko Kanemitsu, Marc-Olivier Sauvain, Didier Trono

Strategies to enhance the capacity of grafted stem/progenitors cells to generate multipotential, proliferative and migrating pools of cells in the postnatal brain could be crucial for structural repair after brain damage. We investigated whether the over-expression of basic fibroblast growth factor 2 (FGF-2) in neural progenitor cells (NPCs) could provide a robust source of migrating NPCs for tissue repair in the rat cerebral cortex. Using live imaging we provide direct evidence that FGF-2 over-expression significantly enhances the migratory capacity of grafted NPCs in complex 3D structures, such as cortical slices. Furthermore, we show that the migratory as well as proliferative properties of FGF-2 over-expressing NPCs are maintained after in vivo transplantation. Importantly, after transplantation into a neonatal ischaemic cortex, FGF-2 over-expressing NPCs efficiently invade the injured cortex and generate an increased pool of immature neurons available for brain repair. Differentiation of progenitor cells into immature neurons was correlated with a gradual down-regulation of the FGF-2 transgene. These results reveal an important role for FGF-2 in regulating NPCs functions when interacting with the host tissue and offer a potential strategy to generate a robust source of migrating and immature progenitors for repairing a neonatal ischaemic cortex.

Oxford University Press2007

Assessing the role of Hes1 and identification of target genes in human and murine T-ALL

Silvia Wirth

The Notch signalling pathway is an ancient cell signalling mechanism that enables short-range communications between cells and controls a broad spectrum of cell fates and developmental processes. In the haematopoietic system Notch signalling has been linked to physiological, but also to pathological phenotypes of T cell development. Identification of numerous activating mutations within the NOTCH1 receptor in human T cell acute lymphoblastic leukaemia (T-ALL) patient samples demonstrated that aberrant activation of this signalling pathway is a frequent event in T-ALL. Active Notch signalling leads to the expression of a plethora of genes that might be drivers or passengers in tumorigenesis including Hes1, which encodes a transcriptional repressor. We have therefore studied Hes1 in the context of T-ALL in mouse models that mimic human disease and in human T-ALL cell lines with the aim to evaluate whether Hes1 affects disease development and disease maintenance and to characterize its function on a molecular level. The first part of this study is dedicated to the functional characterisation of Hes1 in vivo in T-ALL mouse models and in vitro in human T-ALL cell lines. Using a retroviral bone marrow transplant model of T-ALL in which progenitor cells are transduced with a retrovirus expressing constitutive active Notch1 intracellular domain (NICD) we could show that Hes1 is not required in established, late stage T-ALL cells, but that it promotes tumour progression in the early stages of the disease. We have also investigated the function of HES1 in human T-ALL and can show that the overexpression of dominant negative versions of Hes1 in two patient derived cell lines (T-ALL1, CUTLL-1) does not affect proliferation or apoptosis, emphasising that HES1 does not affect cell viability once cells are fully transformed to the leukemic state. In the second part of the study we set out to systematically delineate the gene regulatory networks down-stream of Hes1 by combining RNA-seq and ChIP-seq analysis. Comparing the gene expression of murine premalignant, early stage NICD induced CD4+CD8+ DP cells in the presence and absence of Hes1 by RNA-seq, we unexpectedly found a predominant upregulation of genes in cells expressing Hes1 indicating that Hes1 might rather act as a positive regulator of transcription than as a transcriptional repressor of distinct target genes. Hes1 might influence transcription through secondary or indirect effects as combinatorial analysis of RNA-seq and ChIP-seq led to the identification of only a small number of potential direct target genes that are transcriptionally regulated by Hes1. In summary, the role of Hes1 in promoting the early stages of the disease appears to be highly complex and is likely to involve both direct and indirect control of gene regulatory networks.