We have been developing tools and resources that make it possible to analyze a large number of genes in various experimental conditions. In our earlier work, we 1) constructed cDNA libraries from early mouse embryos and stem cells and generated a large number of expressed sequence tags (ESTs), 2) developed a glass-slide microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 44,000 unique mouse transcripts, 3) produced the web-based ANOVA-FDR software to provide user-friendly microarray data analysis, and 4) developed an algorithm and a fully-automated computational pipeline for transcript assembly from expressed sequences aligned to the mouse genome. In addition, we developed a comprehensive database and web browser of the binding sites of transcription factors (TFs) and cis-regulatory modules (CRMs) on the mouse genome. These resources and tools have then been applied to the systematic analysis of gene regulatory networks in mouse embryonic stem cells. Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. We have demonstrated that it is possible to analyze and identify downstream target genes by monitoring the global gene expression patterns of mouse ES cell lines, when a gene encoding a specific TF is manipulated so that the gene can be overexpressed or repressed. Previously, we generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this NIA Mouse ESC Bank, we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. In further detail, we inferred an increased proportion of cells with neural progenitor marker PSA-NCAM after induction of several TFs. We identified early activation of the Notch signaling pathway as a common feature of most potent inducers of neural differentiation. The majority of neuron-like cells generated by induction of Ascl1, Smad7, Nr2f1, Dlx2, Dlx4, Nr2f2, Barhl2, and Lhx1 were GABA-positive and expressed other markers of GABAergic neurons. In the same way, we identified Lmx1a and Nr4a2 as inducers for neurons bearing dopaminergic markers and Isl1, Fezf2, and St18 for cholinergic motor neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific messenger RNA (mRNA) and microRNAs (miRNAs). Sets of Ascl1-induced mRNAs and miRNAs were enriched in Ascl1 targets. In additional studies, enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using microbeads resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and expressed markers of GABAergic neurons. We also explored the balance between seemingly antagonistic effects of RA on ESCs: differentiation and support of pluripotency. Although ESCs indeed differentiated in the presence of LIF after RA treatment, colonies of undifferentiated ESCs eventually emerged from these differentiated cells even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies after transfer to the standard ESC medium retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.
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