107One of the key advances in this area has been the use of exon-based microarrays, where individual transcript compo-

nents are associated with a unique probe set. Alternate splicing of mRNA transcripts constitutes an important source of gene product diversity across multiple cell types, and the more uniform probe representation afforded by the exon array format allows both the quantitation of differential gene expression, and the identification of particular transcript isoforms present. We can therefore measure the expression of particular splice variants across different cell popula- tions, as well as the changes in exon usage within messages expressed during specific cellular transitions.

Using this combination of technologies we are now able to resolve numerous regulatory binding events affecting not only gene expression, but splice variation across the genome. The advantages of this technique become particularly evident when transcriptional status is measured over time, as differential exon usage can then be observed in response to varying promoter occupancy (figure 1). This analysis is greatly enabled by direct programmatic access to Ensembl (Flicek et al., 2008), facilitating the accurate integration of current genome annotation data.

Transcriptional regulation of neural stem cell differentiation

Diva Tommei and Kairi Tammoja, in collaboration with Steven Pollard and Austin Smith, Wellcome Trust Centre for Stem Cell Research, University of Cambridge, and Peter Dirks, University of Toronto

One of the principle cell lines we study are neural stem cells, which can either be converted from ES cells or derived from fetal forebrain tissue. In feeder- and serum-free culture conditions, ES cell self-renewal is maintained by expo- sure to leukaemia inhibitory factor (LIF) and bone morphogenic protein (BMP) in the culture media. Differentiation is blocked by LIF through LIF-receptor/GP130 signalling and STAT3 activation, and by BMP via SMAD-mediated Id signalling. Upon withdrawal of LIF and BMP, ES cells begin to differentiate; lineage selection is determined by specific culture conditions and the introduction of various inductive cytokines. In basal media, spontaneous ES cell differentiation is driven by the ERK signalling pathway, activated in response to autocrine production of fibroblast growth factor 4 (FGF-4).

When ES cells are differentiated in this manner, lineage selection is predominantly neuroepithelial and results in the emergence of a large fraction (50-80%) of Sox1-positive neural precursors. A reporter cell line in which the open reading frame of Sox1 is replaced with eGFP is used to monitor neuroepithelial differentiation, and the expression of a variety of other differentiation markers can be detected in the same manner. Subsequent application of fibroblast growth factor 2 (FGF-2), in combination with epithelial growth factor (EGF), supports the expansion of a clonogenic population of neural progenitor cells that over several passages acquire homogeneous morphology and immunological reactivity, and exhibit characteristic stem cell properties.

Specifically, these neural stem (NS) cells divide indefinitely in culture, exhibit a stable karyotype and retain neuronal multipotency (Glaser et al., 2007). Even after greater than 100 passages, NS cells can differentiate into all three major cell types of the nervous system (neurons, astrocytes and oligodendrocytes) and demonstrate electrophysiological activity (Conti et al., 2005). NS cells lose Sox1 expression but uniformly express the neuronal marker Sox2 and the intermediate filament nestin, undergo proliferation and expansion in the presence of FGF-2 and EGF, and continu- ously self-renew by symmetrical division.

NS cells are morphologically similar to radial glia, the developmental precursors of neurons and glial cells, and display common genetic and surface markers including RC2, Lex1, Pax6, GLAST and brain lipid binding protein (BLBP), among others (figure 2). Immunological identification and isolation of homogeneous ES and NS populations by fluorescence-activated cell sorting (FACS), using markers such as LeX/CD15 (SSEA1), is therefore efficient and robust. Preliminary microarray analysis of FACS-selected ES and NS cell populations has been performed, revealing distinct transcriptional profiles that comprise a multitude of differentially-expressed genes.

The combined ES/NS system constitutes a reproducible and well-defined model of ex vivo stem cell differentiation. Previous studies have reported the identification of neural stem cells from neurospheres, used as a vehicle to proliferate the stem cell population in suspension. In contrast to suspension cultures that comprise a heterogeneous population of cells – some in self-renewal and others exiting the cell cycle and committing to differentiative lineage selection – NS cells are cultured as a stably proliferating monolayer of adherent cells, permitting straightforward maintenance, immunological identification and sorting/selection.

Importantly, both ES and NS cells exhibit strong morphological and behavioural similarities to in vivo cell types (cells of the inner cell mass and radial glia, respectively). The progression of ES and NS cellular differentiation events is likely to be a useful in vitro model of early development. Thus, ES cell conversion to NS cells and differentiated neurons and glia provides an unlimited cellular resource to study cell commitment, fate choice and differentiation within the developing mammalian nervous system.

A related collection of cell lines have also been derived from human glioma multiforme tumour samples. Gliomas are driven by subpopulation of cancer stem cells which display striking similarities to normal NS cells. These glioma neural stem (GNS) cells have been isolated and expanded using the same culture conditions previously used for the

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establishment of NS cells. The normal and diseased counterparts are morphologically and immunohistologically indis- tinguishable, and yet the differentiation behaviour of the cancer stem cells is clearly aberrant.

We are now applying high-throughput transcriptome sequencing to define the comprehensive transcriptional status of stem cells during neural lineage commitment and differentiation to neurons and oligodendrocytes, an event which is positively correlated with patient survival rates in cases of glioblastoma multiforme. This involves whole-transcrip- tome shotgun sequencing to provide unbiased quantitation of coding and non-coding transcripts. During this project we will also analyse both GNS and NS cell populations using a combination of real-time assays and LNA microarrays to identify microRNAs whose transcriptional status is altered in the disease versus normal cell states.

Functional characterisation of non-coding RNAs in neural system development

Pär Engström, in collaboration with Ramesh Pillai, EMBL Grenoble

Eukaryotic gene expression is modulated at many layers of regulatory control. It is becoming apparent that differen- tiation and development involves the action of numerous regulatory non-protein coding RNAs (ncRNAs). We are therefore establishing computational resources for the study of ncRNAs, and conducting experiments to investigate their expression and function during the development of the mammalian central nervous system.

To identify ncRNAs involved in this process, we are using custom microarrays and strand-specific sequencing proto- cols to measure ncRNA expression in the developing mouse brain. To further prioritise ncRNAs that are likely to be functional, we make use of the large number of sequenced genomes to distinguish ncRNAs that have been conserved during evolution. In the absence of a general model for the molecular function of long ncRNAs, we are considering evolutionary conservation at three different levels: structure, sequence and expression. Genome-wide searches for structurally conserved ncRNAs are facilitated by recent algorithmic innovations (Washietl et al., 2007). For RNAs that are neither conserved in sequence nor in structure, the act of transcription itself can serve a regulatory role (Martens

et al., 2004, Hirota et al., 2008). Orthologous RNAs that lack sequence and structure conservation can be identified by

making use of the extensive cDNA and EST collections available for human and mouse (Engström et al., 2006). Characterisation of novel RNAs includes targeted amplification using RACE PCR to determine precise transcription sites, followed by reciprocal overexpression and knockdown studies. In the latter case a panel of RNA-interference screens are performed; this entails the design of siRNA sequences specific to each target, introduction via transfection vectors and assessment of delivery efficiency, GFP-monitored siRNA expression and measurement of transcriptional

Resear ch in 2009 – The Bertone Gr oup

Figure 2. Top: Differentiation into neural stem (NS) cells from neural-rosette structures. A) ES cell primary culture, B, C) immunostaining for specific surface markers. Bottom: NS cells express markers characteristic of radial glia, permitting both accurate identification of differentiation stages and efficient FACS selection of homogeneous cell populations for genomic analysis. (Images: Steve Pollard, University of Cambridge; adapted from Conti et al., 2005).

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