Context-Specific Notch Targets there is a significant overlap of 74 up-regulated genes indicating potential direct target genes of Notch1 in mesodermal cells, whereas there is almost no overlap for the down-regulated genes. Thus, positive regulation seems to be the main mechanism of Notch1 gene regulation, whereas the negative regulation observed accounts for indirect effects. doi:10.1371/journal.pone.0011481.g002 To examine the cell context-dependent regulation of Notch target genes systematically, we have performed genome-wide transcriptome analyses of Notch1-induced genes in murine ESC under different cell extrinsic cues and in mesodermal cells. We show that Notch signaling activates expression of genes involved as key factors in cell differentiation, cell cycle control and apoptosis in a highly cell-extrinsic and cell-intrinsic cell-context dependent manner. In addition to the classical immediate Notch downstream genes of the Hes and Hey famliy of transcriptional repressors, we identified several key transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id (inhibitor of DNA binding or differentiation) proteins that are critically involved in lineage decisions as differentially regulated Notch1 target genes. Based on our findings we propose a model for a cell-context dependent regulatory network controlling cell fate that involves integration of Notch and other cell-extrinsic and cell-intrinsic signals to fine-tune the level of expression of positive and negative lineage determinants for timed, cell-context dependent lineage-decisions. Results and Discussion Activation of Notch1IC induces expression of specific target genes in embryonic stem cells and mesodermal cells in a highly cell-intrinsic and cell-extrinsic context dependent manner To identify cell-context dependent Notch target genes during ESC differentiation, we employed a conditional Notch activation system that allows the timed activation of Notch1 signaling by the addition of 4-hydroxy-tamoxifen (OHT) in an in vitro develop- Figure 3. Cell-context specific Notch1 target genes identified by genome wide gene expression arrays. Venn diagram of differentially Notch1 induced genes in ESC to ectoderm (ESCe), ESC to mesoderm (ESCm), or mesodermal cells (Mesoderm). Numbers at the labeling indicate the total number of regulated genes. Numbers in the areas indicate the overlap of the different conditions. All numbers in parentheses show the quantity of negatively regulated genes. doi:10.1371/journal.pone.0011481.g003 mental model system closely resembling in vivo development [14]. Using EB5 ESC carrying an OHT-inducible form of murine Notch1IC (EB5 NERT [14]) or control cell lines carrying the pCAG expression vector without the NERT cDNA (EB5 control), the genome-wide, activated Notch1 induced changes in the expression of genes in ESC cultured in differentiation-inducing conditions favoring either mesodermal (ESCm) or ectodermal (ESCe) differentiation or in mesodermal cells (Mesoderm) were determined (Fig. 1A). Based on earlier studies on the induction of previously identified Notch1 target genes and on the time required for activated Notch1 to induce cell lineages decisions in our OHT- dependent systems [14,16–18], we chose a 4 h induction period with OHT. To further test whether the identified genes are likely direct targets of activated Notch1, we determined mRNAs regulated by Notch1 signaling in the absence of de novo protein synthesis. The efficient mesodermal differentiation and biological activity of Notch1 activation at the time of RNA isolation was confirmed by differentiating the cells on either OP9 cells or collagen IV and activating Notch1 signaling by OHT (Fig. 1B). In line with our recent study [14], activation of Notch1 signaling by the addition of OHT strongly reduced the generation of Flk1+ mesodermal progenitor cells from EB5 NERT ESC (Fig. 1B), while OHT treatment had no effect on the differentiation of EB5 control ESC (Fig. 1B). Pluripotency of ESC and correct differentiation were further confirmed by monitoring the expres- sion of Rex1 (Zfp42), a gene commonly used as a landmark of pluripotency, and the expression of genes indicative of ectoderm, i.e. Pax6, or of mesoderm, i.e. Brachyury (T), respectively (Fig. 1C). First, we analyzed the gene array data by principal component analysis (PCA) that clusters data sets according to their degree of correlation (Fig. 2A and Video S1). PCA shows that samples of each cell population, i.e. ESC (ESCe and ESCm) and mesodermal cells (Mesoderm), cluster together, regardless of the expression of the NERT protein or the presence of OHT. While control (Con.) and NERT expressing ESC cluster closely together, mesodermal cells cluster slightly apart in control (Con.) and NERT expressing cells, probably because kinetics of mesodermal differentiation varies in individual clones. Cells treated with cycloheximide (CHX) cluster separately from both ESC and mesodermal cells, indicating that blocking protein synthesis profoundly alters the gene expression profile. Importantly, OHT did not influence clustering in any of the cell populations, suggesting that neither the addition of OHT nor Notch signaling globally influences gene expression. Next we investigated the changes in gene expression by the induction of Notch1 signaling. Based on a signal log ratio of 1 (at least 2-fold change in gene expression), 401 annotated genes and 64 expressed sequences were regulated in EB5 NERT cells by the addition of OHT (Fig. 2B and Table S1). None of these genes was regulated by OHT in EB5 control cells (Figs. 2B and Table S1), suggesting that these genes are bona fide Notch target genes. In line with its signal transduction mechanism as a transcriptional activator, activated Notch1 mostly up-regulated, rather than down-regulated, the expression of target genes (Fig. 2C). Importantly, the majority of Notch1 regulated genes were unique for the cell type, i.e. for ESC (ESCe and ESCm) and mesodermal cells, respectively, and about half of the Notch1 regulated genes in ESC were specific for the culture condition used, i.e. media favoring either mesodermal or ectodermal PLoS ONE | www.plosone.org 4 July 2010 | Volume 5 | Issue 7 | e11481