Unknown,Transcriptomics,Genomics,Proteomics

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Sequentially acting Sox transcription factors in neural lineage development [microarray]

ABSTRACT: We report sequential binding but unique functions of different Sox transcription factors during distinct stages of neural differentiation We used microarray to examine the molecular function of Sox3 in neural progenitor cells. Over-expression of Sox3 under Nestin-promoter in neural progenitor cells. Cells are transgenic for GFP in Sox1 locus and were FACS sorted to obtain pure populations.

ORGANISM(S): Mus musculus

SUBMITTER: Rickard Sandberg

PROVIDER: E-GEOD-33061 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:It remains controversial whether the routes from differentiated cells to iPSCs are related to the reverse order of normal developmental processes or independent of them. Here, we generated iPSCs from mouse astrocytes by three (Oct3/4, Klf4 and Sox2 (OKS)), two (OK), or four (OKS plus c-Myc) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently upregulated during reprogramming and Sox1-positive cells become iPSCs. The upregulation of Sox1 is essential for OK-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during MEF reprogramming, neither Sox1 upregulation nor an increase in neurogenic potential occurs. Thus, astrocytes are reprogrammed through an NSC-like state, suggesting that reprogramming partially follows the retrograde pathway of normal developmental processes. To investigate the gene expression profile of intermediate-state cells during astrocyte reprogramming, we performed genome-wide gene expression analysis in five samples; starting astrocytes, intermediate-state cells expressing Sox1-GFP, NSCs, iPSCs established from astrocytes, and iPSCs established from MEFs (iPS-MEF-Ng-20D-17) that had previously been reported (Okita, K. et al. Nature 448: 313-317 (2007)). Two (NSCs, iPSCs from astrocytes and MEFs) or three (astrocytes, intermediate-state cells) biological replicates were prepared for microarray samples. Total RNA was extracted with an RNeasy kit (Qiagen). cDNA synthesis and transcriptional amplification were performed using 50-100 ng of total RNA with the GeneChip WT PLUS Reagent Kit (Affymetrix). Fragmented and biotin-labeled cDNA targets were hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix) according to the manufacturerâ??s protocol. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner.

Project description:This experiment is part of the FunGenES project (FunGenES - "Functional Genomics in Embryonic Stem Cells" partially funded by the 6th Framework Programme of the European Union, http://www.fungenes.org). The experiment was conducted at the "Instituto de Medicina Molecular,Faculdade de Medicina de Lisboa", Portugal. Goal of the experiment is the in vitro generation and characterisation of neurons from embryonic stem (ES) cells. This is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages try to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors (NPs) to their terminal differentiation into neurons and glial cells, is required. In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer system [1]. We established improved conditions to routinely produce highly homogeneous cultures of NPs, which display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. We show that the transition to neuronal production is linked to the organization of NPs into neural tube-like rosettes, where these cells divide and give rise to neurons. In order to characterize the global gene activity correlated with each particular stage of neural differentiation, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five synexpression groups that correlate with successive stages in the path from ES cells to neurons. This resulted in the identification of unique gene signatures for each cell stage, predicting in addition some of the molecular pathways associated with the transitions between these stages. Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons.

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Sequentially acting Sox transcription factors in neural lineage development [microarray]

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