Nodal/Activin Signaling in ES Cell Fate Decisions Bottom panel shows fold change in marker expression during SB treatment compared to KSR media supplemented with 1/5000 DMSO vehicle. Error bars show standard error of the mean (s.e.m) for n = 4 replicates. doi:10.1371/journal.pgen.1002130.g002 Conversely, inhibition of the pathway with SB (Figure 2B) led to the upregulation of trophectoderm specific markers including Dlx3, Esx and Hand1 and a less significant induction of extra- embryonic primitive endoderm markers such as Gata4/6 and Pdgfra. Similar results were obtained when the ES cells were treated with recombinant Lefty1 protein for the same period of time (data not shown) suggesting that the trophectoderm induction was specific to low Nodal/Activin signaling. Interestingly there was no induction of mesendodermal markers as in the Activin treatment and instead some of these such as Gsc, Mixl and Fgf8 were strongly downregulated. Together, these results suggest that perturbation of the level of Nodal/Activin signaling and consequently endogenous Smad2 phosphorylation led to an exit from self-renewal in ES cells towards highly divergent cell fate decisions of either mesendoderm or trophectoderm differentiation. To confirm these results, fluorescent immunostaining was carried out to assess the protein markers of trophectodermal and mesendodermal lineages (Figure S2) after differentiation in serum containing media. The cell fates obtained under these conditions are similar to the results from the marker analysis performed in chemically defined conditions. Differentiated cells staining positive for Mixl and Lim1 in the nucleus could be detected in Activin cultures. Similarly, Hand1 and placental Cadherin (P-cad) positive giant cells could also be derived from SB treated ES cells. Control treatments with a low dose of DMSO carrier (1/5000 dilution) contained large populations of ES cells that stained strongly for Oct4 and SSEA-1. These results confirmed that the level of Nodal/Activin signaling is responsible for at least 3 cell fate decisions. The endogenous level of signaling is permissive for self- renewal and maintenance of pluripotency, an increase in signaling leads to the induction of mesendoderm like cells while reduction of signaling results in trophectoderm differentiation. Differential Levels of Nodal/Activin Signaling Regulates the Expression of Distinct Subsets of Target Genes We hypothesized that for divergent differentiation programs to be initiated in ES cells, differential gene expression mediated by pSmad2 transcription would be a pre-requisite, which is in turn dependent on the level of Nodal/Activin signaling. Each discrete signaling threshold should induce an independent and unique transcriptional signature distinct from other thresholds. To determine the genetic targets regulated downstream of Nodal/ Activin signaling and their pattern of expression, microarray analysis was carried out to examine genome-wide gene expression following Activin, DMSO or SB treatments in chemically defined KSR media for 18 hours. No significant changes in gene expression out of 26,000 probes could be detected between the DMSO and KSR media control suggesting that the effect of the low concentration of DMSO was negligible on ES cells (Figure 3A). In contrast, Activin and SB treatments induced specific changes in gene expression compared to the DMSO and KSR media controls. Most significantly, we were able to identify subsets of target genes that were regulated by one signaling level and not the other consistent with our hypothesis of threshold specific target gene regulation. For example, 19 genes including Gdf15, Msmb and Orai3 were consistently upregulated in Activin treated cells while showing no significant changes in SB. In contrast, a larger subset of 131 target genes were specifically up- and down-regulated only in the SB treatment and not in Activin. A core subset of 12 targets was co-regulated by both high and low signaling changing their expression in correlation with the treatment including the known Nodal/Activin target genes Lefty1/2 and Pitx2 that were upregulated by Activin and down- regulated in SB. Interestingly, the number of SB regulated targets significantly exceeds that of Activin targets, suggesting that endogenous Nodal/ Activin signaling in ES cells is high or near saturation levels such that a 2-fold increase in pSmad2 could only induce a smaller subset of genes compared to a 2-fold downregulation. Higher doses of Activin treatments and greater than 2-fold increases in pSmad2 may be required to mirror the strength of SB inhibition providing an explanation for asymmetric up- or downregulation of gene expression during different levels of signaling. Some of the target genes driven by Nodal/Activin signaling were indeed implicated in the mesoderm, endoderm and trophectoderm lineages. Fgf15 plays an important role in the development of cardiac mesoderm [43] and Chst15 is specifically expressed in definitive endoderm in vivo [44] with both targets being upregulated by Activin. For SB treatments, Gata3, Tcfap2c and Igf2 were specifically upregulated. Gata3 is a driver of trophecto- derm development [45,46] while Tcfap2c is expressed specifically in the placenta where it regulates essential ADA expression [47,48] and Igf2 is an imprinted gene that modulates nutrient supply between the placenta and fetus [49,50]. Together these target genes support some of the mesendodermal and trophectodermal differentiation programs that may be initiated at 18 hours after the induction of differential Nodal/Activin signaling. With longer- term graded Nodal/Activin signaling over 6 days differentiation, it is likely that additional target genes reinforcing the specification of both lineages may be brought into play over time. Lefty1, Pitx2, Fgf15 and Spsb1 were validated by RT-PCR (Figure 3B) to be co-regulated target genes of high, medium and low signaling displaying a gradient of expression following the signaling level. Cripto, Bcar3, Nphs1 and Cdh3 were targets that were predominantly downregulated by SB inhibition of signaling showing no significant change during Activin stimulation. Conversely, the ID1/2/3 family of transcriptional repressors and Serping1 are specifically upregulated only by the SB treatment showing no difference in response to either Activin or the DMSO control. Hence we conclude that different thresholds of Nodal/ Activin signaling are indeed able to regulate the expression of specific subsets of target genes providing an important explanation for the establishment of divergent differentiation programs. Phospho-Smad2 Binding to Target Gene Subsets and Regulatory Elements Changes Dynamically Depending on the Level of Nodal/Activin Signaling While whole genome microarrays are able to identify the putative subsets of target genes differentially expressed during specific Nodal/Activin signaling levels, this does not provide a molecular mechanism for how different target genes can be directly regulated by the same pathway at different signaling strengths. To address this question, we examined the recruitment of the pSmad2 transcription factor to target genes after subjecting ES cells to Activin, SB or DMSO control treatments in chemically defined KSR media that produce 2-fold up- and downregulation of Smad2 phosphorylation by 18 hours. ChIP-Seq of pSmad2 was employed to identify where pSmad2 was binding on a whole genome scale in parallel cultures of ES cells under the 3 signaling PLoS Genetics | www.plosgenetics.org 6 June 2011 | Volume 7 | Issue 6 | e1002130