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Defining Early Lineage Specification of Human Embryonic Stem Cells by The DEVELOPMENT AND DISEASE RESEARCH ARTICLE 2969 Development 135, 2969-2979 (2008) doi:10.1242/dev.021121 Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/β- catenin, Activin/Nodal and BMP signaling Tomoyuki Sumi1, Norihiro Tsuneyoshi1,2, Norio Nakatsuji2,3 and Hirofumi Suemori1,* The canonical Wnt/β-catenin signaling has remarkably diverse roles in embryonic development, stem cell self-renewal and cancer progression. Here, we show that stabilized expression of β-catenin perturbed human embryonic stem (hES)-cell self-renewal, such that up to 80% of the hES cells developed into the primitive streak (PS)/mesoderm progenitors, reminiscent of early mammalian embryogenesis. The formation of the PS/mesoderm progenitors essentially depended on the cooperative action of β-catenin together with Activin/Nodal and BMP signaling pathways. Intriguingly, blockade of BMP signaling completely abolished mesoderm generation, and induced a cell fate change towards the anterior PS progenitors. The PI3-kinase/Akt, but not MAPK, signaling pathway had a crucial role in the anterior PS specification, at least in part, by enhancing β-catenin stability. In addition, Activin/Nodal and Wnt/β-catenin signaling synergistically induced the generation and specification of the anterior PS/endoderm. Taken together, our findings clearly demonstrate that the orchestrated balance of Activin/Nodal and BMP signaling defines the cell fate of the nascent PS induced by canonical Wnt/β-catenin signaling in hES cells. KEY WORDS: Primitive streak, Mesoderm, Endoderm, Stem cells, β-Catenin, Wnt INTRODUCTION (Thiery, 2002), elucidation of the molecular mechanisms that trigger During early embryogenesis, gastrulation results in the formation of and promote EMT is important for a better understanding of three definitive germ layers and establishment of the embryonic embryogenesis and tumorigenesis. body plan (Tam and Loebel, 2007). At this stage, undifferentiated The canonical Wnt signaling functions are well established in epiblast cells undergo an epithelial to mesenchymal transition fundamental biological processes (Moon et al., 2004; Tam and (EMT) and migrate through a structure called the primitive streak Loebel, 2007). In early embryogenesis, Wnt/β-catenin signaling (PS) to generate the mesoderm and endoderm. Distinct regions of has pivotal roles in the formation of the PS, mesoderm and the PS induce different subpopulation of mesoderm and endoderm endoderm (Lickert et al., 2002; Tam and Loebel, 2007), and the progenitors (Tam and Loebel, 2007). The anterior PS has the stabilization of β-catenin leads to premature EMT in mouse potential to form the definitive endoderm and anterior mesoderm, embryo (Kemler et al., 2004). Wnt binds to its receptor Frizzled including hepatic endoderm and cardiac mesoderm. The middle and co-receptor Lrp5/6, and increases the level of cytoplasmic and region of PS forms the lateral plate mesoderm; and the most nuclear β-catenin, followed by inhibition of the GSK3-mediated posterior region of PS develops extra-embryonic mesoderm degradation pathway. Upon inhibition of GSK3 activity, stabilized progenitors, which give rise to hematopoietic and vascular cells of β-catenin translocates into the nucleus, where it serves as a co- blood islands. Mesoderm and endoderm generation depends on activator of the Lef/Tcf family of DNA-binding proteins to form successful completion of the EMT and migration away from the PS. active transcriptional complexes for specific target genes (Moon The EMT program is the process by which polarized epithelial cells et al., 2004). There is accumulating evidence that the Wnt/β- are converted into individually motile cells; it occurs not only during catenin signaling pathway also has an important role in stem cell early embryogenesis, but also during tumor progression (Thiery, maintenance and regulation of the cell fate decision in several 2002). The important markers of EMT lose epithelial polarities and stem cell systems, including hematopoietic, epidermal and adherence junctions following the downregulation of E-cadherin, intestinal stem cells (Moon et al., 2004). which is an important cell-adhesion molecule of the apical-basal Embryonic stem (ES) cells possess the remarkable property polarity and intercellular adhesion. Although several signaling of indefinite self-renewal and pluripotency, the ability to pathways involved in the EMT processes have been identified differentiate to all cell types of an organism; they also provide an excellent model system for studying cell fate determination in early mammalian development (Keller, 2005). Multiple signaling pathways, such as those involving growth factors, transcriptional regulators and epigenetic modifiers, have crucial roles in 1Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo- regulating the balance between ES-cell self-renewal and lineage ku, Kyoto 606-8507, Japan. 2Department of Development and Differentiation, commitment (Keller, 2005). It is very important to elucidate Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, molecular mechanisms of the self-renewal and lineage Sakyo-ku, Kyoto 606-8507, Japan. 3Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, 69 Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8507, commitment for efficient production of functional cells required Japan. for use of hES cells in transplantation therapy and drug discovery. Like other stem cell systems, canonical Wnt/β-catenin signaling *Author for correspondence (e-mail: [email protected]) is implicated in mouse ES (mES) cell self-renewal and Accepted 3 July 2008 differentiation, depending on the context (Gadue et al., 2006; Hao DEVELOPMENT 2970 RESEARCH ARTICLE Development 135 (17) et al., 2006; Lindsley et al., 2006), but precise roles of this on an ABI Prism 7500 Real-time PCR system using the PowerSYBR Green signaling in human ES (hES) cells remains controversial (Dravid PCR Master Mix (Applied Biosystems, Foster City, CA). The expression et al., 2005; Sato et al., 2004). value of each gene was normalized against the amount of GAPDH and We report here that the activation of canonical Wnt/β-catenin calculated by the ΔΔCt method. The expression level of each gene in the signaling in hES cells by conditional activation of stabilized β- control sample (vehicle or undifferentiated ES cells) was defined as 1.0. The catenin disrupted hES-cell self-renewal. Rather, the canonical normalized expression values for all control and treated samples were β averaged, and average fold-change was determined. Details of the primers Wnt/ -catenin and BMP signaling pathway in hES cells has used for the semi-quantitative and quantitative PCR can be provided on significant roles in establishing the posterior PS/mesoderm request. progenitors, whereas attenuation of BMP signaling changes the cell fate to the anterior PS/endoderm progenitors. In addition, Activin Western blot and immunofluorescence analysis and Wnt/β-catenin signaling pathways synergistically function in Cell lysates were prepared and subjected to sodium dodecyl sulfate- inducing undifferentiated hES cells to differentiate into the anterior polyacrylamide gel elctrophoresis (SDS-PAGE), followed by western PS/endoderm progenitors. This is the first in vitro model system that blotting as described previously (Sumi et al., 2007). For immunofluorescence analysis, cells plated on the culture slides were fixed consistently recapitulates the human early embryogenesis and that with 3.7% formaldehyde and permeabilized with 0.25% Triton X-100. After enables us to analyze molecular events during the process of early blocking, the cells were incubated with primary antibodies, followed by embryogenesis from the epiblast to the PS formation, followed by incubation with secondary antibodies. Alexa Fluor 488- or 555-conjugated lineage specification into the mesoderm and endoderm. More secondary antibodies were purchased from Invitrogen. Cells were mounted importantly, our findings will also be relevant to directed onto glass slides with Vectashield (Vector Laboratories, Burlingame, CA), differentiation of specific tissue and cells from hES cells. and then analyzed using a BX61 fluorescence microscope (Olympus, Center Valley, PA). Antibodies against the following proteins were used: VEGF R2/KDR (clone#89115), SOX17 and CXCR4 (clone#44717) (R&D MATERIALS AND METHODS Systems); E-cadherin, Smad2/3, GSK3β and β-catenin (BD Biosciences); Activation of β-catenin signaling in hES cells N-cadherin (GC-4, Sigma); ZO-1 (Invitrogen); Nanog (ReproCELL); The ⌬Nβ-cateninER construct, in which the N-terminal 90 amino acids were HNF3β, Oct3/4, Brachyury and ERα (Santa Cruz Biotechnology, Santa deleted, was generated by in-frame insertion into the expression vector Cruz, CA); SSEA3 and SSEA4 (Developmental Hybridoma Bank); TRA- containing the hormone-binding domain of a mutant estrogen receptor 1-60 and TRA-1-81 (Chemicon); Phospho-Smad1 (Ser463/465), Smad1, (Littlewood et al., 1995; Sumi et al., 2007). Cell lines expressing ΔNβ- phospho-Smad2 (Ser465/467), phospho-MAPK (Thr202/Tyr204), MAPK, cateninER were obtained by transfection of hES cell lines KhES-1 and phospho-Akt (Ser473), Akt and phospho-GSK3α/β (Ser21/9) (Cell KhES-3 with ΔNβ-cateninER expression plasmids, followed by puromysin Signaling, Danvers, MA). selection as described
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