Nodal/Activin Signaling in ES Cell Fate Decisions scale shows relative Firefly luciferase levels normalized to the co-transfection pGL4.75 Renilla luciferase control. Error bars provide s.e.m. for n = 8 replicates. (D) Real-time PCR quantitation of endogenous Oct4 expression in ES cells treated for 0 to 48 hours in SB (gray bars) compared to DMSO (white bars) after normalizing to the Ywhaz housekeeping control. Vertical axis shows fold change over the 0 h control in each treatment. Error bars show s.e.m. for n = 8 replicates. (E) Western blot of endogenous Oct4 protein levels in ES cells after treatment with SB or DMSO control in a 0 to 24 hour time course. Pcna was used as a loading control in both conditions. Densitometry plot shows Oct4 protein quantitation after normalizing to the respective Pcna loading control with the relative level at 0 hours for each treatment at 100%. doi:10.1371/journal.pgen.1002130.g007 pathway mainly dampens and attenuates its own signaling via negative feedback and less so by positive feedback loops mediated by Nodal, Cripto and FoxH1. One of the intriguing findings is that extracellular signaling gradients were translated into a gradient of Smad2 phosphoryla- tion that we have now shown to be able to recruit different target genes in a dose-dependent manner. This was possibly achieved by an exchange of transcriptional copartners that permits the shifting of the pSmad2 transcriptional complex to different target gene subsets as suggested by the differential recruitment of non-CAGA motifs and comotifs under each signaling condition. The fact that pSmad2 contains only CAGA sequence binding domains and not transcription activation domains suggest that it is further dependent upon copartners for transcription, binding affinity and specificity. In some cases graded pSmad2 transcription complex binding drives graded target gene response that follows signaling strength with high fidelity. In other cases, the target genes are only regulated and responsive at defined signaling thresholds (Figure 8). The consequence is that a relatively modest stimulation with Activin leading to a physiological 2-fold increase in Smad2 phosphorylation eventually drives mesendodermal differentiation while the reciprocal SB inhibition resulting in a 2- fold decrease of pSmad2 is able to promote trophectoderm cell fates. During this process, the master regulator of pluripotency Oct4 is itself titrated by the same Nodal/Activin signaling gradients in the ES cells undergoing differentiation. Hence the same pathway is able to tilt the balance in favor of maintenance of pluripotency or mediate an exit from self-renewal and entry into a specific lineage program. In conclusion, this study for the first time, to our knowledge, reconciles the multiple divergent roles of Nodal/Activin signaling in both pluripotency and differentiation with pSmad2 playing a central role in the cell fate decision making process. Materials and Methods Cell Culture E14 TG2A mouse embryonic stem cells (ATCC) were propagated in FBS media consisting of 20% ES cell-qualified FBS in DMEM supplemented with 100 mM non-essential amino acids, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM GlutaMAX-I (Invitrogen), 55 mM b-mercaptoethanol (Sigma) and 1X homemade Leukemia inhibitory factor (LIF). For the establishment of Nodal/Activin signaling gradients in chemically defined conditions, KSR media containing 20% Knockout Serum Replacement (KSR, Invitrogen) in place of FBS with all other components of ES media excluding LIF were used. For acute (0 to 48 hours) signaling conditions, 25000 ES cells/cm2 were plated for 18 hours in FBS media followed by adaptation of the cells to chemically defined conditions with 10 mM SB-431542 (Tocris) in KSR media for 6 hours as previously described [41]. High signaling was induced by treatment with KSR media containing 25 ng/ml Activin (R&D Systems) or low signaling with 10 mM SB and maintenance of endogenous signaling with control KSR media or 1/5000 dilution of DMSO vehicle as indicated. For long- term differentiation, 2000 ES cells/cm2 were plated and 18 hours later directly treated with Activin, DMSO and SB in FBS media without LIF or KSR media for 6 days with media change everyday. The DMSO vehicle used to dissolve SB can induce differentiation and loss of pluripotency in ES cells [65,66]. In the microarray analysis of the 3 signaling conditions, the effect of DMSO on differential gene expression was determined by comparing against the unsupplemented KSR media control (Figure 3A). The SB inhibitor was used at a high stock concentration of 50 mM permitting 5000X dilution of DMSO in ES cell cultures which was well below the limit required for differentiation. The cultures and treatments were carried out for the microarray study in 4 biological replicates consisting of ES cells at 4 different passages from P20 to P24 to identify and eliminate any cell culture variation effects from analysis. Western Blotting ES cells were lysed in RIPA buffer (150 mM NaCl, 1% NP- 40, 0.5% Sodium Deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0) for protein extracts. SDS-PAGE was performed on 10% polyacrylamide gels and transferred on Immun-Blot PVDF membranes (Bio-rad Laboratories) followed by probing with 1:1000 dilutions of rabbit anti-Smad2 (pSer465/467, Calbiochem), rabbit anti-Smad2 (Invitrogen), mouse anti-Pcna (Santa Cruz Biotechnology) and goat anti-Oct4 (Santa Cruz Biotechnology). Secondary antibodies used were 1:1000 donkey anti-rabbit IgG- HRP (GE Healthcare), 1:1000 goat anti-mouse IgG-HRP and 1:2500 donkey anti-goat IgG-HRP (Santa Cruz Biotechnology). Densitometry measurements of protein bands on western blots were acquired using Photoshop CS3 (Adobe Systems Incorpo- rated). PCR Quantitation of Gene Expression and ChIP DNA Enrichments For gene expression, total RNA was extracted from cells using the RNeasy Mini kit (Qiagen) as per manufacturer instructions. This was reverse transcribed into cDNA using the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems). Quantitative real-time PCR was performed on the 7900HT Fast Real-Time PCR System (Applied Biosystems) or the Biomark System (Fluidigm Corporation) on cDNA or ChIP DNA according to manufacturer instructions. For RT-PCR, products amplified for 25 to 33 cycles were resolved on a 2.5% agarose gel. Primer sequences for both ChIP-qPCR and gene/marker expression can be found in Table S3. Microarray Analysis Total RNA was reverse transcribed into cDNA and in vitro transcribed into biotin-labeled cRNA using the Illumina Total- Prep RNA Amplification kit (Ambion). This was hybridized on MouseRef-8 v2.0 Expression BeadChips (Illumina). Raw intensity values were subjected to background subtraction on the Bead- Studio Data Analysis Software (Illumina) and normalized using the cross-correlation method [67]. Differential gene expression was identified based on a fold change cutoff of .1.5 compared to the DMSO control. The microarray data was deposited in NCBI GEO with accession number GSE23239. PLoS Genetics | www.plosgenetics.org 15 June 2011 | Volume 7 | Issue 6 | e1002130