been predominantly studied in a binary context where they are either present or absent in a biological system. This view has only been able to account for some of their many and often conflicting roles. Our findings challenge this view and support multi-level signaling in stem cells where different signaling strengths can engender different cell fate decisions reflective of the in vivo development of embryos directed not just by Nodal/Activin signaling but possibly Hedgehog, FGF, Wnt and other morphogen pathways. Results The Peak of Transcriptional Activity Induced by Graded Nodal/Activin Signaling Occurs at 18 Hours The direct cellular function of the Nodal/Activin pathway notably of the downstream components Smad2/3/4 is for the regulation of transcription. To address the relation between graded signaling and how they affect transcription, we quantified the changes in expression of known target genes under different signaling levels in chemically defined KSR media conditions. Pluripotent mouse embryonic stem (ES) cells were used to assess the mechanism of morphogen activity as they can differentiate into all tissue types of the adult and express all components of the pathway permitting response to manipulated Nodal/Activin signaling. Some of the known target genes include Pitx2 and Lefty2 which are responsible for the establishment of left-right asymmetry during early embryogenesis, a key developmental role of Nodal/ Activin signaling [32]. In addition, both Lefty2 and Smad7 function as inhibitors of the pathway in a negative feedback mechanism for the attenuation of Nodal/Activin signaling strength [33,34]. Although direct Smad2/3 binding and regulation of the Pitx2 and Lefty2 genes have not yet been demonstrated, in vivo reporter assays suggest that specific enhancers are responsive to Nodal/ Activin signaling and are active only on the left side of the embryo [35,36]. Moreover, these enhancers have been shown to contain FoxH1 binding sites, a known key transcriptional copartner of Smad2/3. Smad7 has been shown to be a direct target of Smad2/ 3/4 binding in the promoter region by gel shift assays [37,38] and it antagonizes the interaction of Smad2/3 with the Type I kinase receptors [39] during negative feedback. Using real-time PCR quantitation, the expression of the 3 target genes was examined in the ES cells following the induction of high signaling by direct treatment with Activin in a time-course. In the reciprocal experiment, the small chemical inihibitor SB-431542 that specifically prevents the kinase domains of the Type I kinase receptors from phosphorylating Smad2/3 [40] was used to generate low Nodal/Activin signaling conditions. Pitx2, Lefty2 and Smad7 were up- and downregulated in direct correlation with the level of signaling under chemically defined conditions compared to the DMSO carrier control representing endogenous or medium signaling (Figure 1). Over the course of 24 hours, the maximum expression of Pitx2 and Lefty2 occurred at 18 hours (Figure 1A and 1B) while that of Smad7 (Figure 1C) occurred earlier at 12 hours. We therefore conclude based on these known target genes that Nodal/Activin signal transduction and its effects on transcription require up to 18 hours to fully develop and any earlier time points result in weaker inductions. We further confirmed that Pitx2, Lefty2 and Smad7 are direct targets of the Nodal/Activin pathway by conducting chromatin immunoprecipitation of phosphorylated Smad2 in the ES cells under the same chemically defined conditions at 18 hours followed by quantification of the enriched genomic DNA fragments by real- time PCR using tiling primers (Figure S1 and Table S3). The Nodal/Activin Signaling in ES Cell Fate Decisions antibody used for the pulldown was raised against the phosphor- ylated serines 465 and 467 on the carboxy-terminus of Smad2 that are specifically targeted by TGFbeta signaling and not by EGF/ ERK/MAPK signaling. At 18 hours where there is maximum expression of the 3 target genes, there was also a robust divergence in the level of pSmad2 binding according to the signaling level for the enhancers of Pitx2 and Lefty2 (Figure S1A and S1B). Interestingly pSmad2 binding was invariant on the known TGFbeta response element of the Smad7 promoter (Figure S1C). This suggested that Nodal/Activin target genes had different binding efficiencies for pSmad2 at each Nodal/Activin signaling level and this was not uniformly changed for all target genes. In conclusion, we confirm that Pitx2, Lefty2 and Smad7 were direct targets of Nodal/Activin signaling and graded pSmad2 binding. Differential signaling sustained for 18 hours also leads to the maximum level of differential gene expression with clear changes in pSmad2 binding on the Pitx2 and Lefty2 genes. Graded Nodal/Activin Signaling Mediates 3 Distinct Cell Fate Decisions in ES Cells Given the downstream changes in pSmad2 binding and transcription of the known direct target genes, we next addressed how extracellular signal levels are translated into intracellular levels of signal transduction. We hypothesized that this could be directly related to changes in pSmad2 levels in ES cells as a consequence of Type I receptor kinase activity. Hence ES cells subjected to differential morphogen signaling conditions may be able to produce different amounts of pSmad2 in cells generating a corresponding differential level of intracellular signaling that leads to differential transcription. It has recently been shown that overexpression of the constitutively active Alk4 type I kinase receptor is sufficient to drive phosphorylation of Smad2 independent of all other Nodal/ Activin receptor complex components [41]. Here we show that direct treatments of the ES cells with Activin and the specific Type I receptor kinase inhibitor SB-431542 in chemically defined conditions also tightly regulates receptor complex activity and produces the phosphorylation of Smad2 in a signaling dependent manner (Figure 2A). During Activin stimulation (high signaling) for 18 hours, there is a defined 2-fold increase in pSmad2 levels while repression with 10 mM SB (low signaling) leads to a 2-fold decrease that is within the limits of physiological change compared to the DMSO vehicle control (equivalent of medium signaling). Differential signaling had no effect on the equilibrium of total Smad2 suggesting that only phosphorylation and not regulation of the total Smad2 population is mediated by Nodal/Activin signaling. Hence extracellular signaling levels are translated into an equivalent gradient of intracellular Smad2 phosphorylation in ES cells. Subsequently we addressed the long-term consequences of increased and decreased signaling on ES cell fate decisions by examining how manipulation of the pathway recapitulates in vivo cell fate decisions by direct treatment with Activin or SB for 6 days. Analysis of a broad range of early cell fate markers (Figure 2B) shows that enhanced Nodal/Activin signaling promotes mesendoderm differentiation in ES cells with strong upregulation of mesendodermal lineage genes including Gsc, Mixl, Eomes and Fgf8. The marker for mesoderm, Brachyury (T), was also strongly induced although this was not reflected by the other mesodermal markers such as Flk1 and Tbx6. This was consistent with the finding that T is also co-expressed in mesendoderm in vivo at the anterior primitive streak [42]. Taken together, this suggests that high signaling induced by Activin predominantly drives mesendoderm differentiation. PLoS Genetics | www.plosgenetics.org 3 June 2011 | Volume 7 | Issue 6 | e1002130