Signaling In Embryonic Stem Cells Research Articles

Glutamate secretion by embryonic stem cells as an autocrine signal to promote proliferation

Glutamate, the major excitatory neurotransmitter in the central nervous system, has also been found to play a role in embryonic stem (ES) cells. However, the exact mechanism and function of glutamatergic signaling in ES cells remain poorly understood. In this study, we identified a glutamatergic transmission circuit in ES cells that operates through an autocrine mechanism and regulates cell proliferation. We performed biological analyses to identify the key components involved in glutamate biosynthesis, packaging for secretion, reaction, and reuptake in ES cells, including glutaminase, vesicular glutamate transporter, glutamate N-methyl-d-aspartate (NMDA) receptor, and cell membrane excitatory amino-acid transporter (EAAT). We directly quantified the released glutamate signal using microdialysis-high performance liquid chromatography-tandem mass spectrometry (MD–HPLC–MS–MS). Pharmacological inhibition of endogenous glutamate release and the resulting tonic activation of NMDA receptors significantly affected ES cell proliferation, suggesting that ES cells establish a glutamatergic autocrine niche via releasing and responding to the transmitter for their own regulation.

Lnc956 regulates mouse embryonic stem cell differentiation in response to DNA damage in a p53-independent pathway.

Maintaining genomic stability is crucial for embryonic stem cells (ESCs). ESCs with unrepaired DNA damage are eliminated through differentiation and apoptosis. To date, only tumor suppressor p53 is known to be implicated in this quality control process. Here, we identified a p53-independent quality control factor lncRNA NONMMUT028956 (Lnc956 for short) in mouse ESCs. Lnc956 is prevalently expressed in ESCs and regulates the differentiation of ESCs after DNA damage. Mechanistically, Ataxia telangiectasia mutated (ATM) activation drives m6A methylation of Lnc956, which promotes its interaction with Krüppel-like factor 4 (KLF4). Lnc956-KLF4 association sequestrates the KLF4 protein and prevents KLF4's transcriptional regulation on pluripotency. This posttranslational mechanism favors the rapid shutdown of the regulatory circuitry of pluripotency. Thus, ATM signaling in ESCs can activate two pathways mediated by p53 and Lnc956, respectively, which act together to ensure robust differentiation and apoptosis in response to unrepaired DNA damage.

Mucin-type O-glycosylation controls pluripotency in mouse embryonic stem cells via Wnt receptor endocytosis.

Mouse embryonic stem cells (ESCs) can differentiate into a range of cell types during development, and this pluripotency is regulated by various extrinsic and intrinsic factors. Mucin-type O-glycosylation has been suggested to be a potential factor in the control of ESC pluripotency, and is characterized by the addition of N-acetylgalactosamine (GalNAc) to serine or threonine residues of membrane-anchored proteins and secreted proteins. To date, the relationship between mucin-type O-glycosylation and signaling in ESCs remains undefined. Here, we identify the elongation pathway via C1GalT1 that synthesizes T antigen (Galβ1-3GalNAc) as the most prominent among mucin-type O-glycosylation modifications in ESCs. Moreover, we show that mucin-type O-glycosylation on the Wnt signaling receptor frizzled-5 (Fzd5) regulates its endocytosis via galectin-3 binding to T antigen, and that reduction of T antigen results in the exit of the ESCs from pluripotency via canonical Wnt signaling activation. Our findings reveal a novel regulatory mechanism that modulates Wnt signaling and, consequently, ESC pluripotency.This article has an associated First Person interview with the first author of the paper.

H3K18ac Primes Mesendodermal Differentiation upon Nodal Signaling.

SummaryCellular responses to transforming growth factor β (TGF-β) depend on cell context. Here, we explored how TGF-β/nodal signaling crosstalks with the epigenome to promote mesendodermal differentiation. We find that expression of a group of mesendodermal genes depends on both TRIM33 and nodal signaling in embryoid bodies (EBs) but not in embryonic stem cells (ESCs). Only in EBs, TRIM33 binds these genes in the presence of expanded H3K18ac marks. Furthermore, the H3K18ac landscape at mesendodermal genes promotes TRIM33 recruitment. We reveal that HDAC1 binds to active gene promoters and interferes with TRIM33 recruitment to mesendodermal gene promoters. However, the TRIM33-interacting protein p300 deposits H3K18ac and further enhances TRIM33 recruitment. ATAC-seq data demonstrate that TRIM33 primes mesendodermal genes for activation by maintaining chromatin accessibility at their regulatory regions. Altogether, our study suggests that HDAC1 and p300 are key factors linking the epigenome through TRIM33 to the cell context-dependent nodal response during mesendodermal differentiation.

O-GlcNAc on PKCζ Inhibits the FGF4-PKCζ-MEK-ERK1/2 Pathway via Inhibition of PKCζ Phosphorylation in Mouse Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ.

Microprinted Stem Cell Niches Reveal Compounding Effect of Colony Size on Stromal Cells-Mediated Neural Differentiation.

Microenvironmental factors have a major impact on differentiation of embryonic stem cells (ESCs). Here, a novel phenomenon that size of ESC colonies has a significant regulatory role on stromal cells induced differentiation of ESCs to neural cells is reported. Using a robotic cell microprinting technology, defined densities of ESCs are confined within aqueous nanodrops over a layer of supporting stromal cells immersed in a second, immiscible aqueous phase to generate ESC colonies of defined sizes. Temporal protein and gene expression studies demonstrate that larger ESC colonies generate disproportionally more neural cells and longer neurite processes. Unlike previous studies that attribute neural differentiation of ESCs solely to interactions with stromal cells, it is found that increased intercellular signaling of ESCs significantly enhances neural differentiation. This study offers an approach to generate neural cells with improved efficiency for potential use in translational research.

Tumorigenic and Differentiation Potentials of Embryonic Stem Cells Depend on TGFβ Family Signaling: Lessons from Teratocarcinoma Cells Stimulated to Differentiate with Retinoic Acid.

A significant challenge for the development of safe pluripotent stem cell-based therapies is the incomplete in vitro differentiation of the pluripotent stem cells and the presence of residual undifferentiated cells initiating teratoma development after transplantation in recipients. To understand the mechanisms of incomplete differentiation, a comparative study of retinoic acid-induced differentiation of mouse embryonic stem (ES) and teratocarcinoma (EC) cells was conducted. The present study identified differences in proliferative activity, differentiation, and tumorigenic potentials between ES and EC cells. Higher expression of Nanog and Mvh, as well as Activin A and BMP4, was found in undifferentiated ES cells than in EC cells. However, the expression levels of Activin A and BMP4 increased more sharply in the EC cells during retinoic acid-induced differentiation. Stimulation of the Activin/Nodal and BMP signaling cascades and inhibition of the MEK/ERK and PI3K/Act signaling pathways resulted in a significant decrease in the number of Oct4-expressing ES cells and a loss of tumorigenicity, similar to retinoic acid-stimulated EC cells. Thus, this study demonstrates that a differentiation strategy that modulates prodifferentiation and antiproliferative signaling in ES cells may be effective for eliminating tumorigenic cells and may represent a valuable tool for the development of safe stem cell therapeutics.

Dickkopf Wnt signaling pathway inhibitor 1 regulates the differentiation of mouse embryonic stem cells in vitro and in vivo

Embryonic stem cells (ESCs) are pluripotent stem cells derived from early stage embryos. It remains unclear whether inhibiting the Wnt/β-catenin signaling pathway using dickkopf Wnt signaling pathway inhibitor 1 (DKK1) impacts on the differentiation potential of mouse ESCs in vitro and in vivo. In the present study, immunohistochemical staining was used to measure the expression of markers of the three germ layers in ESCs and teratomas derived from ESCs. The expression of markers for the Wnt/β-catenin signaling pathway were detected by reverse transcription-polymerase chain reaction (RT-qPCR). Immunohistochemistry and western blotting indicated that the expression levels of octamer-binding transcription factor 4 in the DKK1-treated ESC group were significantly greater compared with the control ESCs. Reduced expression levels of NeuroD and bone morphogenetic protein 4 were observed in the DKK1-treated ESCs and teratomas derived from DKK1-treated ESCs compared with the control group. Increased expression levels of SOX17 were observed in the DKK1-treated ESCs compared with the control group. RT-qPCR indicated that β-catenin expression was significantly reduced in DKK1-treated ESCs and teratomas derived from DKK1-treated ESCs compared with the control groups. Western blotting indicated no alterations in the expression of GSK-3β, however, the levels of phosphorylated-GSK-3β were significantly greater in the DKK1 treatment groups, while cyclin D1 and c-Myc expression levels were significantly reduced in the DKK1 treatment groups compared with the control groups. These results suggest that inhibiting Wnt signaling in ESCs using DKK1 may promote mouse ESCs to differentiate into endoderm in vitro and in vivo, and suppress the tumorigenicity of ESCs.

EphB4 forward-signaling regulates cardiac progenitor development in mouse ES cells.

ABSTRACTEph receptor (Eph)‐ephrin signaling plays an important role in organ development and tissue regeneration. Bidirectional signaling of EphB4–ephrinB2 regulates cardiovascular development. To assess the role of EphB4–ephrinB2 signaling in cardiac lineage development, we utilized two GFP reporter systems in embryonic stem (ES) cells, in which the GFP transgenes were expressed in Nkx2.5+ cardiac progenitor cells and in α‐MHC+ cardiomyocytes, respectively. We found that both EphB4 and ephrinB2 were expressed in Nkx2.5‐GFP+ cardiac progenitor cells, but not in α‐MHC‐GFP+ cardiomyocytes during cardiac lineage differentiation of ES cells. An antagonist of EphB4, TNYL‐RAW peptides, that block the binding of EphB4 and ephrinB2, impaired cardiac lineage development in ES cells. Inhibition of EphB4–ephrinB2 signaling at different time points during ES cell differentiation demonstrated that the interaction of EphB4 and ephrinB2 was required for the early stage of cardiac lineage development. Forced expression of human full‐length EphB4 or intracellular domain‐truncated EphB4 in EphB4‐null ES cells was established to investigate the role of EphB4‐forward signaling in ES cells. Interestingly, while full‐length EphB4 was able to restore the cardiac lineage development in EphB4‐null ES cells, the truncated EphB4 that lacks the intracellular domain of tyrosine kinase and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular domain is essential for the development of cardiomyocytes. Our study provides evidence that receptor‐kinase‐dependent EphB4‐forward signaling plays a crucial role in the development of cardiac progenitor cells. J. Cell. Biochem. 116: 467–475, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.

Mediator Med23 deficiency enhances neural differentiation of murine embryonic stem cells through modulating BMP signaling.

Unraveling the mechanisms underlying early neural differentiation of embryonic stem cells (ESCs) is crucial to developing cell-based therapies of neurodegenerative diseases. Neural fate acquisition is proposed to be controlled by a 'default' mechanism, for which the molecular regulation is not well understood. In this study, we investigated the functional roles of Mediator Med23 in pluripotency and lineage commitment of murine ESCs. Unexpectedly, we found that, despite the largely unchanged pluripotency and self-renewal of ESCs, Med23 depletion rendered the cells prone to neural differentiation in different differentiation assays. Knockdown of two other Mediator subunits, Med1 and Med15, did not alter the neural differentiation of ESCs. Med15 knockdown selectively inhibited endoderm differentiation, suggesting the specificity of cell fate control by distinctive Mediator subunits. Gene profiling revealed that Med23 depletion attenuated BMP signaling in ESCs. Mechanistically, MED23 modulated Bmp4 expression by controlling the activity of ETS1, which is involved in Bmp4 promoter-enhancer communication. Interestingly, med23 knockdown in zebrafish embryos also enhanced neural development at early embryogenesis, which could be reversed by co-injection of bmp4 mRNA. Taken together, our study reveals an intrinsic, restrictive role of MED23 in early neural development, thus providing new molecular insights for neural fate determination.

MiR-125b Regulates the Early Steps of ESC Differentiation through Dies1 in a TGF-Independent Manner

Over the past few years, it has become evident that the distinctive pattern of miRNA expression seen in embryonic stem cells (ESCs) contributes to important signals in the choice of the cell fate. Thus, the identification of miRNAs and their targets, whose expression is linked to a specific step of differentiation, as well as the modulation of these miRNAs, may prove useful in the learning of how ESC potential is regulated. In this context, we have studied the expression profile of miRNAs during neural differentiation of ESCs. We have found that miR-125b is upregulated in the first steps of neural differentiation of ESCs. This miRNA targets the BMP4 co-receptor, Dies1, and, in turn, regulates the balance between BMP4 and Nodal/Activin signaling. The ectopic expression of miR-125b blocks ESC differentiation at the epiblast stage, and this arrest is rescued by restoring the expression of Dies1. Finally, opposite to miR-125a, whose expression is under the control of the BMP4, miR-125b is not directly regulated by Transforming Growth Factor beta (TGFβ) signals. These results highlight a new important role of miR-125b in the regulation of the transition from ESCs to the epiblast stage and add a new level of control on TGFβ signaling in ESCs.

SCF/KIT Inhibition Has a Cumulative but Reversible Effect on the Self-Renewal of Embryonic Stem Cells and on the Survival of Differentiating Cells

The receptor tyrosine kinase c-KIT is expressed in embryonic stem cells (ESCs) and adult stem cells, and many functional studies have demonstrated the importance of the SCF/KIT signaling pathway in adult stem cell maintenance. In this study, we show that a high level of KIT expression in wild-type ESCs correlates with an enhanced self-renewal and that inhibition of KIT signaling in ESCs for extended periods of time has a cumulative but reversible effect on self-renewal. Together these data suggest that continued KIT signaling in some cells within a self-renewing ESC population is required for optimal ESC function. Using a KIT blocking antibody, we recapitulated the phenotype we previously reported for genetically deficient KIT-null cells, demonstrating that SCF/KIT signaling is essential for the survival of differentiating ESCs. Here we show that this phenotype is also reversible. Pharmacological inhibition of JNK also had a cumulative but reversible detrimental effect on the survival of differentiating cells, thus recapitulating the Kit null phenotype and implicating JNK as a downstream mediator of KIT signaling. In contrast, the self-renewal of ESCs was unaffected by prolonged exposure to the JNK inhibitor, suggesting that JNK-independent downstream pathways are involved in KIT-mediated ESC self-renewal whereas KIT-mediated survival of differentiating ESC is likely to be JNK dependent.

Wnt Signaling Regulates the Lineage Differentiation Potential of Mouse Embryonic Stem Cells through Tcf3 Down-Regulation

Canonical Wnt signaling plays a rate-limiting role in regulating self-renewal and differentiation in mouse embryonic stem cells (ESCs). We have previously shown that mutation in the Apc (adenomatous polyposis coli) tumor suppressor gene constitutively activates Wnt signaling in ESCs and inhibits their capacity to differentiate towards ecto-, meso-, and endodermal lineages. However, the underlying molecular and cellular mechanisms through which Wnt regulates lineage differentiation in mouse ESCs remain to date largely unknown. To this aim, we have derived and studied the gene expression profiles of several Apc-mutant ESC lines encoding for different levels of Wnt signaling activation. We found that down-regulation of Tcf3, a member of the Tcf/Lef family and a key player in the control of self-renewal and pluripotency, represents a specific and primary response to Wnt activation in ESCs. Accordingly, rescuing Tcf3 expression partially restored the neural defects observed in Apc-mutant ESCs, suggesting that Tcf3 down-regulation is a necessary step towards Wnt-mediated suppression of neural differentiation. We found that Tcf3 down-regulation in the context of constitutively active Wnt signaling does not result from promoter DNA methylation but is likely to be caused by a plethora of mechanisms at both the RNA and protein level as shown by the observed decrease in activating histone marks (H3K4me3 and H3-acetylation) and the upregulation of miR-211, a novel Wnt-regulated microRNA that targets Tcf3 and attenuates early neural differentiation in mouse ESCs. Our data show for the first time that Wnt signaling down-regulates Tcf3 expression, possibly at both the transcriptional and post-transcriptional levels, and thus highlight a novel mechanism through which Wnt signaling inhibits neuro-ectodermal lineage differentiation in mouse embryonic stem cells.

Abstract 5439: Developmental checkpoint function of Δ40p53.

Abstract Δ40p53, an N-terminally truncated p53 isoform, is predominantly expressed in embryonic stem cells (ESCs) and during early post-implantation embryogenesis in the mouse. We have discovered that Δ40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs. Our studies revealed this isoform of p53 inhibits retinoic acid-induced differentiation and subsequent epithelial-mesenchymal transition (EMT). As ESCs differentiate, Δ40p53 cannot activate the expression of some important developmental genes, such as p21, FGF10, DKK1, lamin A and ZO-1, coincident with decreasing Erk signaling and an enhanced Wnt/β-catenin pathway. ChIP assays suggested that p21, FGF10 and ZO-1 are inhibited at the elongation step. To distinguish between the transcription regulation functions of full-length p53 and Δ40p53, we generated stable HeLa cell lines transfected with Flag-tagged p53 and Δ40p53. qPCR and ChIP assays confirmed that Δ40p53, but not full-length p53, represses gene elongation, with a concomitant decrease in acetylation of histone H4 and H3 in the promoter region. Our studies reveal that Δ40p53, but not p53, is able to block ESC differentiation by regulation of specific gene expression at the elongation step during mouse development. Citation Format: Piyan Zhang, Heidi Scrable. Developmental checkpoint function of Δ40p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5439. doi:10.1158/1538-7445.AM2013-5439

Wnt Pathway Regulation of Embryonic Stem Cell Self-Renewal

Embryonic stem cells (ESCs) can generate all of the cell types found in the adult organism. Remarkably, they retain this ability even after many cell divisions in vitro, as long as the culture conditions prevent differentiation of the cells. Wnt signaling and β-catenin have been shown to cause strong effects on ESCs both in terms of stimulating the expansion of stem cells and stimulating differentiation toward lineage committed cell types. The varied effects of Wnt signaling in ESCs, alongside the sometimes unconventional mechanisms underlying the effects, have generated a fair amount of controversy and intrigue regarding the role of Wnt signaling in pluripotent stem cells. Insights into the mechanisms of Wnt function in stem cells can be gained by examination of the causes for seemingly opposing effects of Wnt signaling on self-renewal versus differentiation.

Oct4 was a novel target of Wnt signaling pathway

The specific expression of Oct4 during early mouse development is required for the correct maintenance of pluripotent cells, and the regulatory control of the Oct4 expression is important. Wnt signaling could have multiple and/or complex effects on embryonic stem (ES) cells characteristics. Elucidation of the molecular mechanisms affecting Wnt signaling in ES cells could provide a better understanding of how these effects occur. The purpose of this study was to determine whether Oct4 was regulated by Wnt signaling in undifferentiated ES cells. Here, we report Oct4 as a novel target of β-catenin-mediated transcription. First, we observe that Wnt signaling pathway is activated in undifferentiated mouse ES cells. In 239T cells, Oct4 promoter was regulated by β-catenin. Through promoter mapping and chromatin immuno-precipitation assays, we found that Oct4 is a direct target of β-catenin/TCF-mediated transcription and the binding site at -875/-881 of Oct4 promoter is critical for b-catenin/TCF-dependent expression regulation. We further detect the expression of Oct4 in treatment with glycogen syntheses kinase (GSK)-3-specific inhibitor in mouse ES cells and HepG2 cells. We found that GSK-3-specific inhibitor can maintain the expression of Oct4 in ES cells and can enhance the expression of Oct4 in HepG2 cells. Our results suggest that Oct4 might be a novel target of β-catenin/TCF-mediated downstream gene in Wnt-activated cells.

Retinoic acid influences the embryoid body formation in mouse embryonic stem cells by induction of caspase and p38 MAPK/JNK‐mediated apoptosis

Although all-trans retinoic acid (RA), the oxidative metabolite of vitamin A, is essential for normal development, high levels are teratogenic in many species. RA results in immediate effects on the preimplantation embryo and on blastocyst development in vitro and in vivo. To further elucidate the cellular mechanisms of early postimplantation embryo development induced by RA, we present an embryonic cell line, B5, as a candidate system for the investigation of these processes. We used undifferentiated ES cells as the model, which is from the undifferentiated status to differentiated status [embryoid body (EB) formation] mimicking postimplantation embryo development (egg-cylinder stage of embryo formation) to clarify the cellular mechanism of action of RA in the implanted blastocysts and cell apoptosis following the series of exposures to differing RA concentrations. Using an in vitro model, we identified the impact of RA on undifferentiated embryonic stem (ES) cells, including inhibition of cell proliferation and induction of cell apoptosis. JNK, P-38 and caspase activation were shown in the nature of RA-triggered apoptotic signaling in ES cells. The carry-on influences of RA on the ES cell were shown in the formation of EB from the pretreated ES cells. RA resulted in apparent impact on undifferentiated ES cells in vitro, with increased numbers of apoptotic cells initially and inhibited cell proliferation, which led to decreased size of EB. The process of EB formation (mimicking the early postimplantation embryo development) is regulated by RA-induced apoptosis through the activation of caspase and P38 MAPK/JNK pathway.

A Genome-Wide siRNA Screen Identifies Novel Phospho-enzymes Affecting Wnt/β-Catenin Signaling in Mouse Embryonic Stem Cells

The Wnt family of signaling molecules is an important regulator of stem cell biology, affecting many developmental processes, including cell growth and proliferation, cell differentiation, and embryonic patterning [1]. Wnt proteins are secreted, highly glycosylated, ligands that activate cytoplasmic pathways via ligand interaction with the coreceptors; low density lipoprotein receptor related protein 5/6 (LRP5/6) and frizzled G-protein coupled receptors [2]. βcatenin is central to Wnt signaling and when Wnt signaling is silent, forms a destruction complex with Axin, Adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3β), casein kinase 1 (CK1), and Disheveled (Dvl), promoting CK1 and GSK3β-mediated phosphorylation and subsequent degradation of β-catenin [3]. Upon a Wntmediated signal, the destruction complex dissociates, allowing β-catenin transit to the nucleus and interaction with transcription factors of the lymphoid enhancer binding factor (LEF)/T cell factor (TCF) family [4, 5] which triggers downstream expression of genes involved in decreasing cellular adhesion, increasing cell migration and differentiation [6]. β-catenin also plays a central role in the regulation of adherens junctions, linking N-cadherin to α-catenin and the actin cytoskeleton [7]. Wnt/β-catenin signaling is critically important for maintenance of embryonic stem (ES) cell pluripotency and is highly up-regulated in ES cells [8–10]. It plays a crucial role in controlling cell expansion in many types of stem cells [11]. Wnt can also influence cell lineage decisions in certain stem cell types by promoting specific fates at the expense of others [11]. Thus, Wnt signaling elicits multiple functions in stem cells. Wnt activity appears to depend on cell-intrinsic properties that might change with time during development, thereby altering the cellular response to Wnt [11]. However, detailed understanding of kinase/phosphatasedependent pathways involved in the regulation for Wnt signaling in ES cells is still not well understood. With the advent of genome wide RNA-mediated interference (RNAi) screening technologies, it is now possible to define the functional contributions of nearly every gene in the mammalian genome to specific signaling pathways or cellular processes in an unbiased manner [12–14]. In this study, we used ES cells as a platform to complete a more specific screen of mouse kinases and phosphatases that may be involved in the regulation of Wnt/β-catenin signaling.

Δ40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs

Δ40p53 is a transactivation-deficient isoform of the tumor suppressor p53. We discovered that Δ40p53, in addition to being highly expressed in embryonic stem cells (ESCs), is the major p53 isoform during early stages of embryogenesis in the mouse. By altering the dose of Δ40p53 in ESCs, we identified a critical role for this isoform in maintaining the ESC state. Haploinsufficiency for Δ40p53 causes a loss of pluripotency in ESCs and acquisition of a somatic cell cycle, while increased dosage of Δ40p53 prolongs pluripotency and inhibits progression to a more differentiated state. Δ40p53 controls the switch from pluripotent ESCs to differentiated somatic cells by controlling the activity of full-length p53 at critical targets such as Nanog and the IGF-1 receptor (IGF-1R). The IGF axis plays a central role in the switch between pluripotency and differentiation in ESCs-and Δ40p53, by controlling the level of the IGF-1R, acts as a master regulator of this switch. We propose that this is the primary function of Δ40p53 in cells of the early embryo and stem cells, which are the only normal cells in which this isoform is expressed.

Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Members of the transforming growth factor-beta superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-beta pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.