Signaling control of differentiation of embryonic stem cells towards mesendoderm Lu Wang, Ye-Guang Chen PII: DOI: Reference:

S0022-2836(15)00353-8 doi: 10.1016/j.jmb.2015.06.013 YJMBI 64782

To appear in:

Journal of Molecular Biology

Received date: Revised date: Accepted date:

17 February 2015 12 June 2015 17 June 2015

Please cite this article as: Wang, L. & Chen, Y.-G., Signaling control of differentiation of embryonic stem cells towards mesendoderm, Journal of Molecular Biology (2015), doi: 10.1016/j.jmb.2015.06.013

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ACCEPTED MANUSCRIPT Signaling control of differentiation of embryonic stem cells towards mesendoderm

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Lu Wang and Ye-Guang Chen

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*Corresponding authors: Ye-Guang Chen, PhD School of Life Sciences Tsinghua University Beijing 100084 China Tel.: 86-10-62795184 Fax: 86-10-62794376 Email: [email protected]

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The State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China

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ACCEPTED MANUSCRIPT Mesendoderm (ME) refers to the primitive streak in mammalian embryos, which has the ability to further differentiate into mesoderm and endoderm. A better

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understanding of the regulatory networks of mesendoderm differentiation of embryonic stem (ES) cells would provide important insights on early embryo

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patterning and a possible guidance for ES applications in regenerative medicine. Studies of developmental biology and embryology have offered a great deal of knowledge about key signaling pathways involved in primitive streak formation.

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Recently, various chemically-defined recipes have been formulated to induce differentiation of ES cells towards mesendoderm in vitro, which greatly facilitate the

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elucidation of the regulatory mechanisms of different signals involved in ME specification. Among the extrinsic signals, TGF-β/Activin signaling and Wnt signaling have been shown to be the most critical ones. On another side, intrinsic

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epigenetic regulation has been indicated to be important in ME determination. In this review, we summarize the current understanding of the extrinsic and intrinsic

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regulations of ES cells-to-ME differentiation and the crosstalk among them, aiming to

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Key words

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get a general overview on ME specification and primitive streak formation.

Embryonic stem cells; Mesendoderm; Activin; Wnt; Gastrulation; Primitive streak; Histone modification.

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ACCEPTED MANUSCRIPT 1. Introduction

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One of the most important events in embryogenesis is the generation of three germ layers: ectoderm, mesoderm and endoderm during gastrulation of embryo

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development. In mouse, once the oocyte is fertilized with a sperm and forms a zygote, which, after a series of cleavage divisions, progressively develops into morula, a mass of cells (blastomeres). Along with the formation of a cavity inside the

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morula, the embryo develops to blastocyst, with the outer cells becoming trophectoderm and the inner cells forming the inner cell mass (ICM). The ICM is

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composed of the pluripotent cells and the blastocyst interior cells. The pluripotent cells further become epiblast that is the source of three germ layers of gastrula, while the blastocyst interior cells will form the primitive endoderm, which give rise to the

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endoderm layer of extraembryonic tissues. Then gastrulation starts, which is marked by the formation of a transient structure called primitive streak in the region of the

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epiblast; and during this process, uncommitted epiblast cells mobilize, egress to

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form the primitive streak, then quickly exit from the primitive streak and form the mesoderm or defined endoderm [1]. The mesoderm gives rise to bone, heart,

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vascular tissue, muscle and kidney while the endoderm develops to lung, liver, pancreas, stomach and intestine (Figure 1A). The ICM-derived mouse embryonic stem (ES) cells and epiblast-derived human embryonic stem cells have the full potential to differentiate to ectoderm, mesoderm, endoderm and trophoblast. During their differentiation to mesoderm and endoderm, these ES cells go through an intermediate stage called mesendoderm (ME), which is equivalent to the primitive streak [2; 3; 4]. However, different from human ES cells that can be directly induced to differentiate to ME, mouse ES cells need to be induced to form embryoid body (EB) before ME differentiation [5; 6]. Several genes have been shown to specifically express in the primitive streak 3

ACCEPTED MANUSCRIPT and to be required for primitive streak formation (Table 1). For instance, Brachyury (T), which is expressed throughout the primitive streak [7], and loss of function

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studies indicate its essential role in primitive streak formation and ME differentiation [8]. As a T-box transcription factor, Brachyury controls the expression of many

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developmental related genes. Similarly, Mix-like homeodomain protein 1 (MixL1) is also expressed throughout the primitive streak, and its ablation results in failure of primitive streak formation and ME differentiation [9]. But the expression of MixL1

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seems to be delayed and is only detected in later stages of differentiation. In addition, there are other genes marking the primitive streak, such as Fgf8 [10], Wnt3 [11; 12],

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Nodal [13], and Eomesodermin (Eomes) [13]. Consistent with their primitive streak-specific expression patterns in the early embryo, these marker genes are upregulated during the in vitro differentiation of ES cells to ME [14]. As an important

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stage for embryogenesis, molecular analyses and lineage tracing studies have defined the anterior and posterior regions of the primitive streak, which eventually

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develop to endoderm and mesoderm tissues, respectively. These regions differ in

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gene expression patterns and development potentials [15] (Table 1). For instance, Foxa2 is preferentially expressed in the anterior region of the primitive streak [16]

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while Flk-1 is in the posterior region [17; 18]. The formation of the primitive streak, as well as subsequent mesoderm and endoderm, is not random but highly organized and spatiotemporally controlled via the interaction of many intrinsic factors (transcription factors, epigenetic regulators and chromatin remodeling factors) and extrinsic factors [1; 19; 20; 21; 22; 23]. Among the extrinsic growth factors, the transforming growth factor-β (TGF-β) superfamily members Activin/Nodal [24; 25] and bone morphogenetic proteins (BMPs) [26], and Wnt family members [27], are the key regulators of these processes.

Moreover,

substantial

progress

has

been

recently

made

in

understanding the roles of histone modifications and chromatin regulators in ES cell differentiation. Histone modifications, such as H3K4me3, H3K27me3 and H3K9me3, 4

ACCEPTED MANUSCRIPT and chromatin regulators, such as Polycomb group proteins, JMJD3, or UTX, have been demonstrated to play important roles in the primitive streak formation of

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embryos and ME differentiation of ES cells [28; 29; 30; 31]. In this review, we summarize the recent progress of elucidating the regulatory

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networks of ME differentiation of human and mouse ES cells, with focus on signaling regulation, epigenetic control and the crosstalk between extrinsic signals and

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epigenetic regulators.

2. Extrinsic signals regulate mesendoderm differentiation

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Several growth factors-initiated extrinsic signals have been demonstrated to play critical roles in primitive streak formation of mouse embryos and mesendoderm

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differentiation of ES cells. Among them, TGF-β superfamily members, Wnt molecules and fibroblast growth factors (FGFs) are the key regulators, which act in a

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co-operative manner to determine mesendoderm cell fate.

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2.1 TGF-β signaling in mesendoderm differentiation The TGF-β superfamily comprises dozes of secreted growth factors including

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TGF-β, Nodal, Activin, BMPs, growth differentiation factors (GDFs) and others. These secreted cytokines transduce the signals via binding to their cognate pairs of cell surface serine/threonine kinase receptors (type I and type II receptors). Type II receptors-mediated phosphorylation leads to activation of type I receptors, which in turn phosphorylate and thus activate R-Smads (receptor-regulated Smads). Phosphorylated R-Smads then form a complex with the common Smad Smad4 and are accumulated in the nucleus to regulate transcription of target genes [32; 33; 34; 35]. In general, different ligands employ different R-Smads to transduce its signal, with TGF-β, Activin and Nodal utilizing Smad2 and Smad3 while BMPs utilizing Smad1, Smad5 and Smad8. In addition, there is another group of Smad proteins that exert inhibitory effect, Smad6 and Smad7. These I-Smads are upregulated by 5

ACCEPTED MANUSCRIPT TGF-β, activin and BMP, thus functioning as negative feedback regulators of TGF-β/BMP signaling [36; 37] (Figure 2A). It is well documented that the TGF-β

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superfamily members play prominent roles in early vertebrate development in vivo

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as well as mesendoderm differentiation of ES cells in vitro [38; 39; 40].

2.1.1 Activin/Nodal signaling regulates mesendoderm differentiation Activin/Nodal signals have long been established as essential ones in

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gastrulation, and its functional conservation has been demonstrated during evolution [41]. Nodal is expressed in epiblast cells prior to gastrulation and then is confined to

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the posterior side, where the primitive streak is formed [24]. Nodal mutant embryos are arrested at the egg cylinder stage and fail to form the primitive streak [42]. As the key signaling mediator of Activin/Nodal signaling, Smad2 is essential for early

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embryo development as Smad2-deficient mice become abnormally short after implantation and entirely lack tissues of the embryonic germ layers [43; 44; 45; 46].

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In ES cells, Activin/Nodal signaling is essential for ME differentiation, and this

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process is hampered when Activin/Nodal signaling is inhibited by the small molecule SB431542 that blocks Activin/Nodal type I receptor activity [2; 47; 48], or when

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Smad2 is knocked down [49]. Genome-wide RNA-seq and ChIP-seq studies showed that, upon activation of Activin/Nodal signaling, Smad2/3, together with Smad4, bind to the promoters of ME signature genes and induce their transcription [13; 40; 50], such as Brachyury [51], Eomes [13] and MixL1 [52]. Through these ME signature genes, which are transcription factors, signaling is amplified, resulting in the activation of groups of ME related genes and pushing the cells towards the ME cell fate (Figure 2A). A number of transcription co-factors have been reported to interact with Smad2/3 and regulate the recruitment of Smad2/3/4 to the regulatory regions of ME genes. For example, TRIM33 (TIF1, ectodermin), a member of the TIF1 family of transcriptional cofactors, binds Smad2/3, and the resulting complex is recruited to H3K9me3 and 6

ACCEPTED MANUSCRIPT H3K18ac on the promoters of ME-regulating genes Goosecoid (Gsc) and MixL1 [6]. The recruitment of the TRIM33-Smad2/3 complex makes the nodal response

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element accessible to the Smad2/3-Smad4 complex and RNA polymerase II, consequently leading to gene transcription. The H3K27me3 demethylase JMJD3

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has been shown to interact with Smad2/3 upon Activin/Nodal activation and demethylate the H3K27me3 on the Nodal and Brachyury loci marked by the Polycomb group proteins, and the removal of H3K27me3 in turn upregulates the

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expression of Nodal, Brachyury and other ME related genes [53]. The histone acetylation factor p300/CBP is essential for the Smad2/3/4-mediated activation of

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the RNA polymerase II transcription machinery [54], and the chromatin remodeling protein Brg1 helps to sustain a suitable chromatin state for Smad2/3/4-induced transcription [55]. Furthermore, the transcription co-factors FoxH1 and Mixer

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contribute to the binding specificity of Smad2/3 on certain gene loci and selective

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activation of these genes [25; 56] (Figure 2B).

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2.1.2 The role of BMP signaling in mesendoderm differentiation BMP signaling has broad impact on embryo development and ES cells fate

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determination. In mammals, BMPs are involved in specification of nearly all kinds of tissues and organs, including the nervous system, lung, kidney and others [26]. BMP4 is expressed at a low level in the embryo prior to gastrulation and then reaches to the peak in the posterior primitive streak [57; 58; 59; 60]. Ablation of BMP4 results in gastrulation defects in mouse embryos [57; 61]. As core mediators of the BMP signaling pathway, both Smad1 and Smad5 are critical for germ layer formation. Smad1 knockout leads in early embryonic lethality due to defects in visceral endoderm and extraembryonic mesoderm formation [59; 62; 63]. Smad5 ablation results in reduction of mesoderm and impaired angiogenesis in yolk sac [64; 65]. In ES cells, BMP4 signaling also plays important roles in cell fate determination, including self-renewal of mouse ES cells [40; 66; 67; 68], trophoblast and 7

ACCEPTED MANUSCRIPT mesendoderm differentiation of human ES cells and mouse epiblast stem (EpiS) cells [2; 69].

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Unlike its well-known developmental functions, the role of BMP4 signaling in ME differentiation is complex and perhaps indirectly, as blockage of BMP4 signaling

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with the antagonist Noggin, the receptor inhibitor LDN193189 or Smad1/5/8 knockdown, has no obvious interference of ME differentiation [70; 71; 72]. A possibility is that BMP4 acts at the up-stream of the ME differentiation chain by

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co-operating with other signals to instruct the differentiation process (Table 2). For instance, BMP can co-operate with FGF to promote ME differentiation by fine-tuning

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the phosphorylation level of the MAP kinase ERK and modulating the transcription of Dusp6 and Nanog [2]. Although there is evidence for direct regulation of ME signature gene expression by Smad1/5/8, the function of BMP signaling in ME

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specification needs further investigation.

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2.2 Wnt signaling in mesendoderm differentiation

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Wnt proteins are a group of secreted glycoproteins and control a variety of cellular events such as cell proliferation, differentiation, migration and cell polarity,

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and have critical roles in embryogenesis [73]. In accordance with their important developmental and physiological functions, deregulation of Wnt signaling results in abnormalities of both mouse and human embryos and causes an array of human diseases including cancer [74]. The canonical Wnt signaling is initiated via the ligand binding to the cell surface seven-pass transmembrane receptor Frizzled and the coreceptor lipoprotein receptor related protein 5/6 (LRP5/6), leading to the recruitment of Axin to Frizzled and Dishevelled to LRP5/6 and disassembly of the β-catenin destruction complex (consisting of Axin, Dishevelled, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1) and β-transducin repeats-containing protein (β-Trcp)) and subsequently the accumulation of β-catenin [75; 76]. Then the stabilized β-catenin enters the nucleus 8

ACCEPTED MANUSCRIPT and complexes with T cell factor (TCF) to activate gene expression [73; 77; 78]. In addition to the canonical Wnt signaling, a group of Wnt ligands can signal without

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LRP5/6 and β-catenin to activate small GTPases (Rac, Rho and Cdc42), c-Jun N-terminal kinase (JNK), calcium/protein kinase C and other molecules, all of which

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are referred as noncanonical Wnt signaling [78; 79].

During mouse embryo development, a number of Wnt proteins are expressed in the ICM or the trophectoderm [80]. β-Catenin exhibits a relatively high expression

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in both the ICM stage of the embryo and throughout the primitive streak, suggesting an important role of the canonical Wnt signaling in gastrulation and primitive streak

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formation [81; 82; 83; 84]. Among several canonical Wnt genes, Wnt3 is expressed throughout the primitive streak, and loss of function of Wnt3 results in an inhibition of gastrulation and loss of the primitive streak [11; 12]. In ES cells, activation of Wnt

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signaling promotes differentiation towards ME and its derivative tissues [85; 86]. Accordingly, interference of Wnt signaling by the antagonist DKK1 or the small

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molecule IWR1-endo dramatically decrease the efficiency of ME differentiation [87;

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88]. The promoting effect of Wnt/β-catenin signaling on ME differentiation seems to be achieved by β-catenin/TCF-mediated upregulation of ME signature genes, such

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as MixL1 [52], Brachyury [51; 89; 90] (Figure 3). Wnt4, Wnt5a and Wnt11, all of which signal through the non-canonical Wnt signaling pathways, have been shown to play important roles during embryogenesis. For example, studies in mice revealed that Wnt11 is required for convergent extension movements during gastrulation [91; 92; 93]. But unlike canonical Wnt signals, the expression of these Wnt ligands was not detected in the primitive streak region of mouse embryos, indicating a less essential role of non-canonical Wnt signaling during primitive streak formation [94]. In ES cells, both Wnt5a and Wnt11 have been reported to function in the late stage of hematopoietic differentiation and cardiac differentiation [94; 95], the processes that need to go through ME. However, these Wnt ligands seem to have little effect in ME differentiation. 9

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2.3 FGF signaling in mesendoderm differentiation

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FGF signaling is important for gastrulation of mouse embryos [96], as the loss-of-function of FGF receptor 1 (Fgfr1) causes mouse lethality at gastrulation and

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defects in mesoderm patterning [97; 98; 99]. Chimeric mouse embryo analysis showed that FGFR1 is required for epiblast cells to traverse the primitive streak [100]. Among the 18 vertebrate FGF genes, Fgf3 [101], Fgf4 [102], Fgf8 [103], and Fgf17

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[104] have been detected in prestreak- and streak-stage of embryos, but only Fgf8-null embryos fail to form the primitive streak and exhibit defective gastrulation

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[10]. The function of FGF signaling in ME differentiation of ES cells has been well documented. FGF2 promotes ME differentiation together with BMP4, Activin A, or

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Wnt signals in a dose-dependent manner [2; 47; 105] (Table 2). The effects of FGF signaling in ME induction and primitive streak formation could be multifaceted:

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orchestrating E-cadherin expression and promoting epithelial-mesenchymal

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transition and cell migration [96].

2.4 Interplay between different signal pathways during ME specification

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ES cells provide an ideal in vitro model for investigation of the molecular mechanisms

underlying

primitive

streak

formation/ME

differentiation.

As

summarized in Table 2, in chemical-defined ES cell differentiation conditions, Activin A at a high dose alone, together with FGF2 and the phosphoinosital-3 kinase (PI3K) inhibitor LY294002 or with serum, can induce Brachyury-positive ME and subsequently differentiation of Sox17-positive defined endoderm [47; 106; 107; 108]. A high dose of Wnt3a alone or BMP4 together with FGF2 or with LY294002 can also induce Brachyury-positive ME as well as the subsequent development of Flk-1-positive mesoderm [2; 17; 18; 70; 81; 82; 109; 110; 111]. In addition, BMP4 alone can induce Brachyury-positive ME and hCGa-positive trophoblast cell fate [2; 70]. 10

ACCEPTED MANUSCRIPT Both Activin A and Wnt3a accelerate ME formation, whereas inhibition of either Activin or Wnt signaling blocks this process [2; 87; 88; 112]. These observations

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suggest that Activin and Wnt specify ME in a co-operative manner (Figure 5). The mechanisms underlying this collaboration recently started to emerge due to detailed

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biochemical analysis and large-scale ChIP-seq, RNA-seq and mass spectrometry approaches [40; 49; 113]. A study from Stephen Dalton’s group showed that PI3K/Akt can switch the function of Activin/Smad2/3 signaling between ES cell

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self-renewal and ME differentiation [48]. In a high level of PI3K/Akt signaling, Activin signals via Smad2/3 promotes ES cell self-renewal by upregulating Nanog and

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suppressing Raf/MEK/ERK signaling and canonical Wnt/β-catenin signaling, both of which have been reported to induce differentiation [114; 115]. When PI3K/Akt activity is decreased, the Wnt effector β-catenin is accumulated and collaborates with

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Smad2/3 to induce MixL1 expression and thus to promote ME differentiation [48]. Recently, Funa et al reported that β-catenin collaborates with Smad2/3 and Oct4 to

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regulate ME differentiation of human ES cells [90]. They found that activation of Wnt

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signaling with the GSK3β inhibitor CHIR99021 could up-regulate the expression of ME signature genes Brachyury, MixL1 and Eomes, and this up-regulation depended

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on β-catenin and Smad2, both of which bind to the same regulatory regions of ME genes. CHIR99021 could also increase Nodal expression, which forms a positive regulatory loop to push ME differentiation. OCT4 is also important in the ME gene expression by co-occupying β-catenin binding regions. Interesting, when Smad2/3 activation is suppressed, β-catenin pushes neural crest differentiation, enforcing the importance of Nodal/Smad signaling in ME differentiation. Another study recently also demonstrated the importance of crosstalk between Wnt and Activin signaling in ME differentiation, but proposed a different mechanism [116]. Wnt3a stimulates the recruitment of RNA polymerase II to the promoters of the ME signature genes via β-catenin/LEF-1, and Activin/Smad2 signaling subsequently increase transcription elongation at these genes, leading to transcription of ME genes. 11

ACCEPTED MANUSCRIPT As the major downstream effectors of Hippo signaling [117], YAP and its paralogue TAZ have been demonstrated to be important in embryogenesis. TAZ-null

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mice are unable to develop to adulthood, and deletion of YAP leads to embryonic lethal by E8.5 owing to yolk sac defects [118; 119]. Jeffrey L. Wrana and his

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colleagues reported a crosstalk between TGF-β signaling and Hippo signaling to balance the pluripotency vs. ME differentiation of ES cells [113]. In ES cells, YAP/TAZ and TEAD maintain the pluripotency by forming a complex with Smad2/3

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and Oct4 and suppressing the expression of differentiation genes and modulating the levels of core pluripotency genes. Upon induction of ME differentiation, the

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YAP/TAZ-TEAD-Smad2/3-Oct complex is disrupted, and Smad2/3 then interact with the ME specifier FOXH1, resulting in upregulation of the ME gene Eomes and driving ME specification [113]. YAP also functions together with TEAD to negatively

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regulate Smad2/3-mediated transcription elongation and then blocks ME genes

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induced by Wnt and Activin [116].

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3. Epigenetic control of mesendoderm differentiation Mesendoderm differentiation of ES cells is not only orchestrated by extrinsic

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cues, the proper states of chromatin and histone modifications are also a prerequisite for transcription activation and differentiation initiation towards ME. In ES cells, many differentiation genes are marked at a “poised" state with the bivalent histone modifications of H3K4me3 (an active mark for transcription) and H3K27me3 (a repressive mark for transcription), ready to be activated in response to differentiation signals [22; 23]. Recently, large-scale ChIP-seq and MethylC-seq analyses indicate that certain kinds of histone modifications, such as histone acetylation including H3K4ac, H3K27ac and H3K9ac and histone methylation including H3K4me3, H3K27me3 and H3K9me3, are linked with specific differentiation stages towards mesenchymal differentiation, neural progenitor induction, trophoblast-like cell differentiation or mesendoderm and subsequent 12

ACCEPTED MANUSCRIPT pancreatic differentiation [30; 31; 120; 121]. Specifically, H3K27me3, H3K4me3 and H3K9ac were suggested to play the most important roles in ME differentiation by

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blocking the expression of ME-related genes, such as Gsc, Brachyury, Sox17 and Eomes in response to extrinsic signals such as BMP4, FGF and Activin [30; 31]

Chromatin

regulators,

including

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(Figure 4). DNA

cytosine

methyltransferases,

methyl-CpG-binding proteins, histone modification enzymes and ATP-dependent

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remodeling complexes, have been reported to function in embryogenesis and ES cell fate determination [122]. During gastrulation, several chromatin regulators have

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been implicated to play essential roles, and elimination of their expression leads to severe phenotypes. For instance, ablation of the chromatin remodelers Brg1, Lsh and Snf5 leads to peri-implantation lethality due to defects of primitive ectoderm and

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trophectoderm [69; 123; 124]. Polycomb Repressive Complex 2 (PRC2), which mediates H3K27me3 modification, is one of the most extensively studied chromatin

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regulators during gastrulation and ME differentiation of ES cells. Embryos lacking

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EZH2, the histone methyltransferase catalytic subunit of PRC2, can initiate gastrulation, but cannot complete the process and form the primitive streak [29].

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Deletion of the core components of PRC2, SUZ12 and EED, yields the similar phenotypes [125; 126; 127]. Large-scale RNA-seq and ChIP-seq analyses revealed that SUZ12 and EZH2 target key developmental regulators in ES cells by marking a relatively high H3K27me3 level [128; 129]. These key regulators include the HOX cluster genes LHX1 that modulates ME-endoderm differentiation and PAX6 that specify neuroectoderm, the FOX genes that are involved in axial patterning[130], the SOX genes that alter cell-fate specification [131], and the TBX genes that regulate a wide variety of differentiation processes such as gastrulation and early pattern formation [132]. Consistently, depletion of SUZ12 or EED in ES cells disrupts ME-specific differentiation and leads to a random differentiation toward all lineages [133; 134]. In addition, other histone methyltransferases, such as RING1B (a core 13

ACCEPTED MANUSCRIPT component of PRC1 that also mediates H3K27me3 modification), G9A (mediating H3K9me2 modification) and MLL (mediating H3K4me3 modification), have been

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reported to be necessary for gastrulation and early embryogenesis [125; 135; 136; 137].

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Dynamic change of histone modifications provides a possible epigenome environment for ME differentiation. Among different types of histone modifications in development and embryogenesis, H3K27me3, H3K4me3 and H3K9ac have been

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found to be closely related to ME differentiation [30; 31; 120]. During differentiation of pancreatic tissue, a definitive endoderm (DE) tissue derived from mesendoderm,

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H3K4me3 and H3K27me3 modifications have been shown to correlate with a rapid cellular transition from the pluripotent state to ME-DE [31; 120]. The inhibition of JMJD3 significantly reduces the expression of ME-DE related genes, indicating that

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the ME-DE fate specification requires removal of H3K27me3 at early stages of differentiation. LSD1 (H3K4me3 demethytase), JMJD3 and UTX (H3K27me3

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demethytases) have been extensively studied during ES cell differentiation. In

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particular, during ME differentiation, JMJD3 and UTX activities are correlated with the transcriptional activation of differentiation genes [138; 139; 140]. The recruitment

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of JMJD3 by Smad2 onto the promoter regions of ME signature genes (Brachyury and Nodal) also supports an important role of JMJD3 during ME differentiation [141].

4. Crosstalk between extrinsic signals and epigenetic regulations Both extrinsic signals and epigenetic regulations need to be appropriately organized and well coordinated to orchestrate a proper ME differentiation. However, the linker between them is still largely unknown. As mentioned above, during Activin/Nodal-induced ME differentiation, Smad2 recruits JMJD3 onto the promoters of the Nodal and Brachyury genes to remove H3K27me3 and thereby activate their transcription [53; 141]. In response to Wnt signaling, JMJD3 and UTX remove H3K27me3 in the promoter region of Wnt3 and induce its expression [139]. Wnt3 is 14

ACCEPTED MANUSCRIPT both a signature gene of ME and a ligand of Wnt signaling. In addition, depletion of either JMJD3 or UTX delays ME differentiation due to deficient Wnt signaling [139].

[142].

This

is achieved

by the

recruitment

of

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Smad2/3 is important to maintain a proper H3K4me3 on pluripotency and ME genes the

H3K4me3

modifier

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Dpy30-COMPASS complex onto these genes by Smad2/3 and Nanog. Consistent with the essential function in ME specification, Dpy30 deletion leads to reduced expression of Nanog and Oct4 and induction of mesendoderm genes [142]. In ES

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cells, the Hippo signaling mediators TAZ/YAP/TEAD and the Activin/Nodal signaling mediator Smad2 form a complex with Oct4 and function through the co-repressor

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NuRD to inhibit the expression of differentiation genes and thereby maintain ES cell pluripotency [113]. TRIM33, upon Activin/Nodal signaling, interacts with Smad2/3, and the resulting complex binds to the nucleosomes containing histone H3 with

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K4me3-K9me3-K18ac modifications in the promoter regions of Gsc and MixL1. The superior affinity of TRIM33 for H3K9me3-K18ac displaces heterochromatin protein

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1 from the promoters, leading to exposure of the Activin-responsive element,

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expression of Gsc and MixL1 and thus ME differentiation [6; 50] (Figure 5).

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5. Conclusions and Perspective The primitive streak formation during mammalian embryogenesis is regulated by integration of different signaling pathways; among the most important ones are Activin and Wnt signaling. Although we have a reasonable understanding on the regulatory mechanism of ME specification by extrinsic signals and by epigenome modification and chromatin remodeling, the crosstalk between extrinsic signals and intrinsic transcriptional and epigenetic regulations are still unclear at large. For example, how different kinds of extrinsic signals coordinate to regulate intrinsic transcription factors and epigenetic modifications during ME specification is still poorly understood. Therefore, global mapping of the regulatory circuits awaits further investigation. In addition, dynamic analyses of histone modifications have 15

ACCEPTED MANUSCRIPT been mainly focused on relatively late stages of endoderm or mesoderm differentiation, the early molecular events of ME specification, especially the link

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between extrinsic signals and epigenetic regulation to control those early events, are unknown. Furthermore, as ME is a transient but an essential stage of the

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differentiation to mesoderm and endoderm tissues, maintenance of the ME identity in an in vitro culture system would not only facilitate the elucidation of the detailed mechanisms of the differentiation into mesoderm and endoderm tissues, but also

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guide directional differentiation to related tissues, which is critical in regeneration

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medicine.

Acknowledgments

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CE

PT

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This work in YGC Lab is supported by grants from the 973 Program (2011CBA01104) and the National Natural Science Foundation of China (31330049, 31221064). YGC is a Bayer-endowed Chair Professor.

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ACCEPTED MANUSCRIPT Table 1 Signature genes of primitive streak/mesendoderm

Gene

Loss of function effect

ES cells

Mouse

ES cells

differentiation

development

differentiation

Mouse

PT

Expression

development

Reference

Failure of

gastrulation and

Primitive

differentiate into

primitive streak

streak

ME

formation

Fail to

Abnormalities in

Primitive

differentiate into

primitive streak and

streak

ME

node formation

Fail to

Failure of EMT and

Posterior

differentiate into

loss of primitive

primitive streak

ME

streak

MixL1

Eomes

24~48h

24~48h

24~48h

~24h

gastrulation and

differentiate into

loss of primitive

ME

streak

streak

FoxA2

AC

FGF8

48~72h

48~72h

[9]

[13]

[11; 12]

Arrest at egg

Epiblast /

Fail to

cylinder and fail to

gastrulation /

differentiate into

form primitive

primitive streak

ME

streak

CE

Nodal

24~48h

PT

Wnt3

[8]

Inhibition of

Fail to

ED

Primitive

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ury (T)

MA

Brachy

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Fail to

[13]

Failure of

Prestreak- and

gastrulation and

streak-stage

primitive streak

embryos

-

formation

[10]

Normal gastrulation Anterior

and primitive streak

primitive streak

-

formation

[143]

Normal gastrulation Goose coid

Anterior 48~72h

and primitive streak

primitive streak

-

17

formation

[144]

ACCEPTED MANUSCRIPT Table 2 Chemical defined differentiation systems in ES cells Differentiation

Cell Fate determination

Cell Line

Refe

Growth Basal medium

Cell

factor/Che

fate

50% IMDM+ 50% F12 NUT-MIX +

H9, hiPS

Activin A / BMP4 /

DE

xA2/Sox17

H9, mEpiSC hiPS

ME-

Brachyrury-FoxA2/S

[47;

cells

DE

ox17

145]

H9, HSF6, mEpiSC,

ME-

Brachyrury-CDX2/TB

hiPS cells

M

X6

ME-

Brachyrury/MixL1/Eo

DE

mes-Sox17

ME-

Brachyrury/Mixl1-Sox

DE

17/PDX1

LY294002

F12 NUT-MIX + FGF2 /

PVA

LY294002 /

50% IMDM+ 50% F12 NUT-MIX +

ED

BMP4

Activin A /

Activin A

AC

CE

DMEM/F12+N2B27

PT

FGF2

PVA

MA

50% IMDM+ 50%

NU

FGF2 /

H1, HSF6

H1, HSF6

[106]

[81]

[107]

[107]

Nβ-cateninER KhES-1/

BIO

MEM

Nβ-cateninER

DMEM/F12+ N2B27

e

Brachyrury/MixL1-Fo

LY294002 Activin A /

renc

ME-

FGF2 /

PVA

Marker

SC RI

mical

PT

Culture condition

Brachyrury/MixL1/Gs c-FoxF1 [108]

KhES-3/hES-3 Activin A /

ME-

Brachyrury/

β-catenin

DE

MixL1-FoxA2

Nβ-cateninER RPMI 1640 +

Activin A

2%FBS

RPMI 1640 + 0.2%FBS + 2%FBS

Activin A

KhES-1/Nβ-cateninE

ME-

R KhES-3/hES-3

DE

MEhES

18

DE

Brachyrury/GSC-Fox A2/CER1

MixL1-FoxA2/Sox17

[108]

[146]

ACCEPTED MANUSCRIPT BMP4 /

H1, H9, H7

MEM/D

FGF2

Brachyrury-KDR/CD 34

E

[70]

H1, H9, H7 BMP4

ME-T

+ Transferin

PT

Brachyrury/ RPMI 1640 + Insulin

Wnt3-hCGa/

[70]

H1, H9, H7 Activin A

NU

H1, H9, H14 BMP4 mTeSR(-FGF)

SC RI

CDX2

ED

FGF2

MA

H1, H9, H14

BMP4 /

WA01, WA07, WA09, BG01, BG02

Brachyrury

DE

-FoxA2/GSC

ME-T

Brachyrury -hCGa

ME-

Brachyrury

M/D

/MixL1-FoxF1

E

/Sox17

ME

Brachyrury /MixL1/Eomes

[70]

[2]

[2]

[52]

PT

Activin A

ME-

CE

HAI

AC

BIO

RIMP1640+B27

mTeSR (full)

WA01, WA07, WA09, BG01, BG02

ME

/MixL1/Eomes

[52]

Wnt3a

H1, H9

ME

Brachyrury

[110]

Activin A

H1, H9

ME

Brachyrury

[110]

1m

Shef1, Shef3

ME

Brachyrury /Gsc

[109]

Shef1, Shef3

ME

Brachyrury /Gsc

Activin A / 1m

H1, H9, Advanced RPMI

Brachyrury

CHIR99021

CHB8-H2B-GFP-hE

ME-

SCs,

M

hiPS cells,

[109]

Brachyrury /MixL1-PAX2/LHX1-

[111]

SIX2/SALL1

1m: GSK3β inhibitor; BIO: GSK3β inhibitor; CHIR99021: GSK3β inhibitor; LY294002: PI3K inhibitor. DE: defined endoderm; E: endoderm; M: mesoderm; ME: mesendoderm; T: 19

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI

PT

trophoblast.

20

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI

PT

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ACCEPTED MANUSCRIPT Figure 1. Primitive streak formation during mouse embryogenesis and mesendoderm differentiation of embryonic stem cells

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(a) When a mouse embryo develops to about embryonic day 3.5, it forms a blastocyst, containing the outer blastomeres that are the precursors of trophoblast

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cells, and the inner blastomeres that form inner cell mass (ICM). The ICM is further differentiated into pluripotent epiblast cells and the cells that contribute to the primitive endoderm. At about E5.5 day, gastrulation begins: epiblast cells form a

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transient structure-primitive streak. The anterior part of the primitive streak eventually develops endoderm tissues such as pancreas, lung and liver, while the

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posterior part forms mesoderm tissues such as bone, vascular tissue and muscle. (b) ES cells derived from the ICM have the potential to form ectoderm, mesoderm, endoderm and trophoblast. Along with the differentiation, ES cells become epiblast

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cells, which have the capability to differentiate to all layers except the trophoblast. Epiblast cells can further differentiate to mesendoderm, which is equivalent to the

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primitive streak, and subsequent mesoderm and endoderm tissues.

Figure 2. TGF-β signaling in mesendoderm differentiation

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(a) The binding of the TGF-β superfamily members Activin and BMP with their cognate serine/threonine kinase receptors (type I and type II receptors) leads to the C-terminal phosphorylation of R-Smads (Smad2/3 for Activin signaling and Smad1/5/8 for BMP signaling). Phosphorylated R-Smads form a complex with Smad4, and the resulting Smad complex is accumulated in the nucleus and together with other transcription cofactors such as p300 and FoxH1 and regulates the expression of mesendoderm genes Brachyury (T), MixL1, and Eomes. Smad6/7 are negative feedback regulators of TGF-β signaling. (b) During ME specification, Smad2 interacts with a number of transcription cofactors to control the expression of ME genes. Among these cofactors, TRIM33 modulates transcription by binding to H3K18ac and H3K9me3, JMJD3 reduces 31

ACCEPTED MANUSCRIPT H3K27me3, p300/CBP promotes acetylation of histones, and FoxH1 helps form

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transcription activation machinery.

Figure 3. The canonical Wnt signaling in mesendoderm differentiation

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Wnt3a binds to the transmembrane receptor Frizzled and co-receptor LRP5/6, leading to the interaction of Dvl with Frizzled and Axin with LRP5/6 and the disassembly of the β-catenin destruction complex. Inhibition of β-catenin

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degradation results in its accumulation in the nucleus and its interaction with TCF3/4

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to activate the transcription of ME genes, such as Brachyury (T) and MixL1.

Figure 4. Epigenetic regulation in mesendoderm differentiation In ES cells, many differentiation-related genes are marked at a “poised" state with

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H3K4me3 (an active mark for gene expression) and H3K27me3 (a repressive mark for gene expression). H3K4me3 is mainly maintained by MLL, while H3K27me3

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maintained by the PRC2 complex containing EZH2, EED and SUZ12. The

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expression of the differentiation genes is repressed in ES cells. Upon induction of ME differentiation, the landscape of histone modifications is changed as the

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H3K27me3 level is reduced due to the decreased activity of the PRC2 complex or its dissociation from the promoters of ME genes, or due to the increased activity of the demethytases JMJD3 and UTX. Reduction of H3K27me3 activates the transcription of the ME genes. Meanwhile, histone acetylation, such as H3K9ac, promotes the expression of the ME genes.

Figure 5. Integration of multiple signaling pathways in mesendoderm differentiation Both Activin and Wnt signaling play critical roles in ME differentiation. Activin functions through Smad2/3/4 and Wnt functions through β-catenin/TCF3/4. Other ME-promoting signals, such as BMP4 through Smad1/5/8/4 and FGF through 32

ACCEPTED MANUSCRIPT PI3K/ERK, seem to function closely with Activin and Wnt signals. Smad2/3/4 activates transcription of the pluripotent gene Nanog and, together with FGF2,

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promotes self-renewal. Upon induction of ME differentiation by extrinsic signals, such as Wnt3a, Activin A, FGF2 and BMP4, these extrinsic signals integrate at the

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chromatin level, together with epigenetic regulators and chromatin remodeling factors, alter the landscape of epigenetic modifications. By collaborating with transcription factors or co-factors, the altered epigenetic modifications favor the

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expression of ME-related genes. For instance, Smad2/3 cooperates with β-catenin and recruits JMJD3 to remove H3K27me3, leading to activation of ME signature

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genes and ME specification.

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Regulatory mechanisms of different signals are involved in mesendoderm specification TGF-β/Activin and Wnt signaling are the most critical extrinsic signals for mesendoderm specification Intrinsic epigenetic regulation is important in mesendoderm determination Current understanding of the extrinsic and intrinsic regulations of ES cells-to-ME differentiation and the crosstalk among them is summarized here

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Signaling Control of Differentiation of Embryonic Stem Cells toward Mesendoderm.

Mesendoderm (ME) refers to the primitive streak in mammalian embryos, which has the ability to further differentiate into mesoderm and endoderm. A bet...
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