Context-Specific Notch Targets Figure 5. Induction heat maps of Notch1 target genes involved in regulation of metabolic process, cell adhesion and cell communication. The identified Notch1 targets were grouped into different Gene Ontology (GO) terms using the DAVID functional annotation analysis tool (http://david.abcc.ncifcrf.gov/). Note that only differentially regulated genes by Notch1 are shown as Log2 transformed values. If no gene symbol was available for the regulated transcript, GenBank accession number was given in parentheses. Significant changes of differentially regulated gene expression according to the criteria outlined in Material and Methods were labeled with a black or white dot for up- and down- regulated genes, respectively. Redundant probe sets for the same gene were removed except when significantly different results were obtained (e.g. Sox9). The complete list of regulated genes is available in the Supplement Table S1. doi:10.1371/journal.pone.0011481.g005 (Fig. 4, 7, 8D). Hence, Sox9 appears to be a direct target of Notch1. Recently, Notch has been shown to bind to the Sox9 proximal promoter in liver cells [22], further supporting a direct link between Sox9 and Notch1. Furthermore, suppression of the Notch1 induced Sox9 upregulation using specific siRNA reverted Notch1 induction of chrondogenesis (S.M., R.S., U.J., R. Haller, J. Kramer, J. Rohwedel, manuscript in preparation), demonstrating Sox9 as critical mediator of Notch-induced cell lineage decisions. Taken together, the direct regulation of Sox9 by activated Notch1 may play an important role for the development of cells in all three germ layers. The neuro-ectodermal transcription factor Pax6 is induced by Notch1 signaling in ESC but not in mesodermal cells Recently, we and others have shown that the activation of Notch has a decisive role in ES cell fate determination [14,15]: Notch suppresses the generation of mesodermal cells from ES cells and promotes neural commitment of ES cells, when cultured in the absence of self renewal and serum factors, suggesting a role for Notch during the specification of the germ layers during mammalian embryogenesis. How Notch mediates this cell lineage specification at the molecular level is currently unknown. Here we have identified the neuroectodermal transcriptional regulator Pax6 as a Notch1 target gene in ESC but not in mesodermal cells (Fig. 4, 7, 8E). Moreover, the observed up-regulation of Pax6 by activated Notch1 was most likely direct in that the up-regulation was not affected by CHX and, thus, independent of protein synthesis (Fig. 4, 7, 8E). The effect of cell-context dependent Notch regulation was further paralleled by Pax6 protein levels (Fig. 10A). Pax6 is crucial for various developmental processes in the central nervous system and other ectodermal tissues [23]. Depending on the cellular context it promotes cell proliferation and expansion of neural stem cells, or neuronal differentiation. Importantly, expression of Pax6 in ESC favors neuroectodermal lineage choice and radial glia formation [24], in a similar way to activated Notch1. Furthermore, in Drosophila and Xenopus, Notch signaling also induces eye-related gene expression, including Pax6 [25]. Taken together, it is thus conceivable that the direct activation of Pax6 expression is involved in Notch-induced differentiation of ESC into radial glia in a context-dependent manner. The mesodermal transcription factor Runx1 is induced by Notch1 signaling in mesodermal cells but not in ESC Runx genes are key regulators of lineage-specific gene expression in major developmental pathways. The expression of Runx genes is tightly regulated, leading to distinct tissue- and developmental- specific expression patterns [26]. Here we identified Runx1 (AML1) as a Notch1 target gene in mesodermal cells but not in ESC (Fig. 4, 7, 8F). Because Runx1 was induced by activated Notch1 in the presence of CHX, i.e. without protein synthesis, Runx1 is likely to be a direct target gene of Notch (Fig. 4, 7, 8F). Runx1 determines commitment of mesodermal progenitor cells to the hematopoietic lineage [27]. In line with our findings, studies in Drosophila, zebrafish and mouse have defined a Notch-Runx1 pathway to be critical for developmental specification of hematopoietic stem cell fate and homeostasis of hematopoietic stem cell number [28–30]. However, although these studies clearly established the depen- dency of Notch signaling on Runx1 up-regulation for hematopoi- etic stem cell fate and maintenance, neither study showed direct transcriptional regulation of Runx1 by activated Notch1. Taken together, we propose that activated Notch influences mesodermal progenitor cells to adopt a hematopoietic stem cell fate by a mechanism involving the cell-context dependent transcriptional up-regulation of Runx1 gene expression. The myogenic transcription factor Myf5 is induced by Notch1 signaling in mesodermal cells but not in ESC Notch signaling plays an important role in the maintenance of muscle progenitor cells during embryogenesis and in the generation and maintenance of satellite cells, the stem cells of mature muscle, in fetal development as well as during regeneration in the adult [31]. The myogenic program is controlled by the combinatorial activity of myogenic regulatory factors comprising Myf5, Myod1, Myogenin and MRF4/Myf6/Herculin which, together with paired domain transcription factors Pax3 and Pax7, are essential for skeletal myogenesis [32]. In RBP-J or Delta1 deficient mice, muscle progenitor cells show premature differen- tiation, leading to depletion of the progenitor pool [33,34]. Since it has been shown that Delta/Notch/RBP-J signaling represses the expression of the Myod1 via activation of Hes1, thereby blocking muscle differentiation [35], and that Myod1 deficiency leads to increased survival of muscle stem cells [36], one way by which Notch signaling maintains the muscle stem cell pool may be by the down-regulation of Myod1. In our screen for Notch target genes, however, we have identified Myf5 as a potential direct Notch1 target gene in mesodermal cells (Fig. 4, 7, 8G). Myf5 is expressed first in the paraxial mesoderm and later in skeletal muscle progenitor cells, and is followed by the expression of Myod1. Myf5 and Myod1 determine two genetically and lineally distinct populations of muscle progenitor cells, which can substitute for each other within the developing embryo [37,38]. Myf5 supports efficient skeletal muscle regeneration by promoting satellite cell proliferation [39] and may be involved in the expansion of the progenitor cell pool. Considering the roles of Myf5 and Myod1 for muscle development, we propose that the direct induction of Myf5 expression together with Hes1-mediated down-regulation of Myod1 by Notch signaling may contribute to the maintenance of muscle progenitor cells. Along this line, the up-regulation of Myf5 and down-regulation of Myod1 by ligand-activated Notch signaling has been demonstrated in C2C12 myoblasts [40]. Thus, there may be several cooperating mechanisms by which Notch signaling maintains the muscle progenitor pool during embryogenesis and in the adult. Transcriptional repressors of the Id family are differentially regulated by Notch1 signaling in a cell- context dependent manner In addition to lineage specific transcription factors we found members of the Id family of bHLH transcriptional repressors to be PLoS ONE | www.plosone.org 8 July 2010 | Volume 5 | Issue 7 | e11481