Context-Specific Notch Targets Figure 8. Regulatory transcriptions factors involved in differentiation are induced by activated Notch1 in the absence of protein synthesis. (A–H) The microarray results were validated by qPCR in ESC cultured in self renewal (ESCs), ectodermal (ESCe), or mesodermal (ESCm) culture medium and in mesodermal cells (Mes.). All conditions were tested in the absence (2) or presence (+) of CHX. Relative expression is shown for untreated (blue bars) and OHT treated samples (red bars) compared to Gapdh. The calculated inductions are indicated by green squares. The error bars indicate the standard deviation from 2 to 6 independent experiments for the cells carrying the inducible Notch1 (NERT). Myf5 (G) is expressed at a very low level in undifferentiated cells (ESCs, ESCe, ESCm). Calculated inductions that do not reach significance above background levels (grey area) are indicated by an asterisk. doi:10.1371/journal.pone.0011481.g008 occupancy and irrespective of active repression by RBP-J or not, the binding of NotchIC to RBP-J mediates the transcriptional switch to activate gene expresssion from target promoters. How RBP-J and NotchIC binding to RBP-J-specific sequences at target gene promoters to activate gene expression is regulated in a cell-context dependent manner is an interesting issue for further investigation (see model below). In our second approach we used knock-in reporter cell lines to confirm target gene activation by Notch1. Using ESC with a lacZ insertion in one myf5 allele [49] or ESC with a Venus gene, a modified version of yellow fluorescent protein, in the Runx1 locus [50], we generated stable ESC lines (Myf5-lacZ- NERT and Runx1-Venus-NERT), which express NERT, an OHT-inducible form of murine Notch1IC [14], or control cell lines carrying the pCAG expression vector without the NERT cDNA (Myf5-LacZ and Runx1-Venus). Functionality of Notch1 pathway activation in Myf5-LacZ-NERT ESC and Runx1-Venus-NERT ESC was tested in undifferentiated ESC by analysis of Hes5 and HeyL expression after culturing the cells for 4 h in the presence or absence of OHT. All Myf5- LacZ-NERT ESC and Runx1-Venus-NERT ESC clones used for further study upregulated Hes5 and HeyL expression in the presence of OHT, while in control clones Hes5 and HeyL expression remained unchanged by the addition of OHT (data not shown). Using embryoid body culture, ESC were differentiated in the presence or absence of OHT to induce Notch signaling and analyzed at different times after plating. Venus expression was significantly induced by the addition of OHT in Runx1-Venus-NERT cells but not in Runx1-Venus control cells (Fig. 10B) at day 5 of mesodermal differentiation. Quantitative FACS analysis of mesodermal progenitor cells expressing Flk1 confirmed mesodermal differentiation and Notch activation, as previously described [14] (Fig. 10B). Similarly, activation of Notch signaling by the addition of OHT strongly induced the expression of b-Gal-positive cells in Myf5-lacZ-NERT cells but not in Myf5-LacZ control cells after 5 days of differentiation (Fig. 10C). Taken together, these results further confirm these genes to be regulated by activated Notch1. A mechanistic model for the role of Notch signaling in stem cell differentiation: Activated Notch promotes lineage entry by activating lineage specific transcription factors and expansion of lineage specific stem and progenitor cells by a negative feed back loop involving Hes/Hey and Id transcriptional repressors What is the consequence of Notch pathway activation in various cell types? Expression of constitutive active Notch promotes the generation of cell lineage specific stem cells and inhibits further differentiation [4]. We have shown here that activated Notch1 most likely directly induces lineage specific transcription factors (LSTFs), which are known to direct multipotent stem cells along a certain lineage and are required for differentiation, in a cell context dependent manner. We further found that Notch1 signaling at the same time also induced Notch target genes of the Hes/Hey family, which can block differentiation by negatively regulating expression and/or function of cell LSTFs, e.g. MyoD or Runx [51]. In addition we observed, that inhibiting protein synthesis augmented the transcriptional up-regulation of direct Notch target genes, indicating the presence of a negative feedback loop requiring the synthesis of a repressive protein. In this respect, the reported binding of Hes/Hey to RBP-J on their own promoters to inhibit their Notch-induced expression [52], suggests that Hes/Hey proteins play a critical role in this negative feedback loop. Taken together, we propose a model (Fig. 12) for the mechanistic function of Notch signaling in stem cell differentiation: Notch signaling activates the transcription of LSTF as well as Hes/Hey proteins. Specific chromatin marks or other transcription factors yet to be identified may determine cell context dependency for differential NotchIC binding. The LSTFs then prime the cells for differentiation along the respective lineages, while the concom- itant up-regulation of Hes/Hey genes counteracts further differ- entiation by interfering with expression or function of these LSTFs. Recently, a similar stimulation of the expression of a gene as well as the repressor of that gene by activated Notch was shown in Drosophila muscle progenitor cells [7]. Thus, activated Notch may promote lineage entry by activating LSTFs and expansion of lineage specific stem and progenitor cells by a negative feed back loop involving Hes/Hey transcriptional repressors. Another layer of complexity as well as a link to other signaling pathways is provided by the differential regulation of Id proteins by activated Notch (Fig. 12A). During differentiation, Id proteins act as a short-lived brake withholding predetermined cells from differentiating, by associating with LSTFs and preventing them from binding DNA. At the same time, Id proteins can bind, possibly cell-context dependently, also to specific Hes proteins, thereby inhibiting the autorepression of certain Hes genes [53]. Thus, the cells are primed to adopt a ’pre-differentiation state’ by the expression of LSTFs and held in check by opposing signals because they express inhibitors of the Id and Hes/Hey family at the same time. Further differentiation will then depend on the availability of LSTFs, which is regulated by the cell-context dependent amount and type of LSTFs, Id and Hes proteins (Fig. 12A). In addition to Notch signaling, the expression of Id proteins is positively regulated by TGFb/BMP signaling in different tissues [41], contributing to the balance of transcription factor availability (Fig. 12B). Similarly, Hes and Hey proteins transduce and integrate signals from TGFb/BMP, JAK-STAT, Ras and HIF signaling pathways [51]. On the other hand, LSTFs are also subject to regulation by several external signaling pathways, such as FGF, TGFb/BMPs and Wnts (Fig. 12B). Furthermore, a positive feed-forward loop up-regulating Notch expression itself may ensure definitive determination into a specific lineage (Fig. 12B). Finally, work in Drosophila, zebrafish and C. elegans has demonstrated that miRNAs are direct transcriptional target genes of Notch signaling and regulate Notch target genes [54–56]. It is thus tempting to speculate, that miRNAs induced by activated Notch further influence the amount of certain transcription factors present and thus PLoS ONE | www.plosone.org 12 July 2010 | Volume 5 | Issue 7 | e11481