Figure 7. Notch1 target genes validated by qPCR. The microarray results were validated by qPCR using ESCs, ESCe, ESCm and Mesoderm. All conditions were tested also in the absence or presence of cycloheximide (+CHX). Oct4 (last gene; also known as Pou5f1) is included as negative non- regulated control. (A) Mean values of relative expression (% of Gapdh expression, Log2 transformed) shown as a heatmap in the absence (2) or presence (+) of OHT. (B) Inductions by activated Notch1 were calculated from relative expression values and were Log2 transformed. Thus, blue areas indicate down-regulation and red regions show up-regulation. *) indicates low level expression of Myf5 near detection limit resulting in artificially high induction values that are not reliable. doi:10.1371/journal.pone.0011481.g007 Context-Specific Notch Targets signaling pathway [42]. Along this line, Notch signaling has been shown to positively regulate the transcription of XId3, a Xenopus member of Id proteins, through a Su(H)-dependent pathway [43]. In this study we found several members of the Id family to be differentially regulated by activated Notch1: While Id1, Id3 and Id4 were up-regulated, Id2 was down-regulated by activated Notch1 (Fig. 4, 7). The significance of this differential regulation is not clear. Id1, Id2 and Id3 show a similar expression pattern during embryogenesis, while the distribution of Id4 is distinct [44]. Due to functional redundancy among the four members of the Id family and their widespread, overlapping expression patterns, only crosses between mice that lack different Id genes to generate multiple knock-outs are embryonic lethal. Id factors have been implicated to play an essential role mostly during early development, in neural development, hematopoiesis and angio- genesis. With regard to Notch signaling, it is of interest to note that Id proteins are short-lived proteins that have been reported to function as intracellular timer of differentiation in the nervous system [44]. It is thus conceivable that Notch upregulates certain Id proteins, in particular Id4 (Fig. 10A), a crucial regulator of neural stem cell fate determination, to control timing of differentiation (see model below). Genetic complementation assays and knock-in reporter cell lines confirm Notch1 target genes identified in the Notch1-induced transcriptome To confirm that the Notch1 target genes identified in our screen are regulated also under physiological conditions in vivo we used two approaches: First, we used genetic complementation of ESC that lack RBP-J [45], a transcription factor that plays a central role for canonical Notch signal transduction. To this end, expression levels of the target genes Hes1, Hes5, Hey1, Hey2, Id4, Pax6, and Sox9 that were likely regulated directly by activated Notch in ESC were analyzed in ESC clones that lacked RBP-J (RBP-J-/-) or had reduced expression of RBP-J (RBP-J+/-), as well as in RBP-J-/- ESC clones that were complemented with RBP-J (RBP-J rescue) or in RBP-J-/- ESC that expressed VP16- RBP-J, a transcriptionally active derivative of RBP-J, from a transgene (VP16-RBP-J rescue) [45]. As expected from the role of RBP-J as transcriptional repressor in the absence of Notch signaling, all three RBP-J-/- ESC clones analyzed expressed significantly higher amounts of Hes1, Hes5, Hey1, Hey2, Id4 and Pax6 (Fig. 11A). For all of these genes, re-expression of RBP-J from a transgene restored the repression (Fig. 11B). In line with active Notch signaling, expression levels for all of these genes were further increased in the presence of the transcriptionally active derivative of RBP-J (Fig. 11B), thus corroborating our array and qPCR data on Notch target genes. However, the regulation of Sox9 by Notch signaling appears to be more complex: Although activated Notch1 upregulated Sox9 expression, we did not observe significantly altered expression levels of Sox9 in ESC lacking RBP-J (Fig. 11A). One possibility for the unaltered expression levels of Sox9 in RBP- J-/- ESC could be that Sox9 is not suppressed by RBP-J, although it is regulated by Notch1 signaling. In the mammalian skin as well as during T helper cell differentiation, target repression by RBP-J does not play an important role [46,47], suggesting that in these tissues Notch signals in an RBP-J independent manner. However, since the expression levels were influenced in some but not all RBP-J-/- ESC clones after re-expressing RBP-J or VP16-RBP-J from a transgene (Fig. 11B), a more likely explanation is that repression of Sox9 by RBP-J is dynamic and depends on other additional factors. Notably, in Drosophila, RBP-J occupancy on many Notch target promoters is a transient, dynamic process [48]. In both, Drosophila and mammalian cells, the presence of NotchIC enhances RBP-J PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11481