Graded Nodal/Activin Signaling Titrates Conversion of Quantitative Phospho-Smad2 Levels into Qualitative Embryonic Stem Cell Fate Decisions

Nodal and Activin are morphogens of the TGFbeta superfamily of signaling molecules that direct differential cell fate decisions in a dose- and distance-dependent manner. During early embryonic development the Nodal/Activin pathway is responsible for the specification of mesoderm, endoderm, node, and mesendoderm. In contradiction to this drive towards cellular differentiation, the pathway also plays important roles in the maintenance of self-renewal and pluripotency in embryonic and epiblast stem cells. The molecular basis behind stem cell interpretation of Nodal/Activin signaling gradients and the undertaking of disparate cell fate decisions remains poorly understood. Here, we show that any perturbation of endogenous signaling levels in mouse embryonic stem cells leads to their exit from self-renewal towards divergent differentiation programs. Increasing Nodal signals above basal levels by direct stimulation with Activin promotes differentiation towards the mesendodermal lineages while repression of signaling with the specific Nodal/Activin receptor inhibitor SB431542 induces trophectodermal differentiation. To address how quantitative Nodal/Activin signals are translated qualitatively into distinct cell fates decisions, we performed chromatin immunoprecipitation of phospho-Smad2, the primary downstream transcriptional factor of the Nodal/Activin pathway, followed by massively parallel sequencing, and show that phospho-Smad2 binds to and regulates distinct subsets of target genes in a dose-dependent manner. Crucially, Nodal/Activin signaling directly controls the Oct4 master regulator of pluripotency by graded phospho-Smad2 binding in the promoter region. Hence stem cells interpret and carry out differential Nodal/Activin signaling instructions via a corresponding gradient of Smad2 phosphorylation that selectively titrates self-renewal against alternative differentiation programs by direct regulation of distinct target gene subsets and Oct4 expression.

[1]  M. Kessel,et al.  FGF8 functions in the specification of the right body side of the chick , 1999, Current Biology.

[2]  Tetsuya Tabata,et al.  Morphogens, their identification and regulation , 2004, Development.

[3]  C. Wernstedt,et al.  Phosphorylation of Ser465 and Ser467 in the C Terminus of Smad2 Mediates Interaction with Smad4 and Is Required for Transforming Growth Factor-β Signaling* , 1997, The Journal of Biological Chemistry.

[4]  J. Rossant,et al.  Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2 , 2010, Development.

[5]  K. Luo,et al.  Negative Feedback Regulation of TGF-β Signaling by the SnoN Oncoprotein , 1999 .

[6]  Chad H. Koonce,et al.  Mice exclusively expressing the short isoform of Smad2 develop normally and are viable and fertile. , 2005, Genes & development.

[7]  A. Hart,et al.  Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo. , 2002, Development.

[8]  A. Brivanlou,et al.  The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development. , 2001, Genes & development.

[9]  A. Schier,et al.  erratum: The zebrafish Nodal signal Squint functions as a morphogen , 2001, Nature.

[10]  D. He,et al.  Transforming growth factor beta -inducible independent binding of SMAD to the Smad7 promoter. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Y. Saijoh,et al.  Distinct transcriptional regulatory mechanisms underlie left-right asymmetric expression of lefty-1 and lefty-2. , 1999, Genes & development.

[12]  J. Miyazaki,et al.  Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells , 2000, Nature Genetics.

[13]  R. Behringer,et al.  Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks. , 2002, Developmental cell.

[14]  F. Conlon,et al.  A primary requirement for nodal in the formation and maintenance of the primitive streak in the mouse. , 1994, Development.

[15]  M. Trotter,et al.  Derivation of pluripotent epiblast stem cells from mammalian embryos , 2007, Nature.

[16]  J. Gauthier,et al.  A short amino-acid sequence in MH1 domain is responsible for functional differences between Smad2 and Smad3 , 1999, Oncogene.

[17]  James Briscoe,et al.  The interpretation of morphogen gradients , 2006, Development.

[18]  J. Belo,et al.  N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase expression during early mouse embryonic development. , 2006, International Journal of Developmental Biology.

[19]  J. Walsh,et al.  Arkadia enhances nodal-related signalling to induce mesendoderm , 2001, Nature.

[20]  F. Lallemand,et al.  c-Jun Associates with the Oncoprotein Ski and Suppresses Smad2 Transcriptional Activity* , 2002, The Journal of Biological Chemistry.

[21]  A. Reith,et al.  SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. , 2002, Molecular pharmacology.

[22]  W. Vale,et al.  Cripto Is a Noncompetitive Activin Antagonist That Forms Analogous Signaling Complexes with Activin and Nodal* , 2008, Journal of Biological Chemistry.

[23]  M. Bitzer,et al.  Smad3 and Smad4 Mediate Transcriptional Activation of the Human Smad7 Promoter by Transforming Growth Factor β* , 2000, The Journal of Biological Chemistry.

[24]  T Hartung,et al.  The effects of solvents on embryonic stem cell differentiation. , 2006, Toxicology in vitro : an international journal published in association with BIBRA.

[25]  J. Massagué,et al.  Mechanisms of TGF-β Signaling from Cell Membrane to the Nucleus , 2003, Cell.

[26]  W. Reik,et al.  Placental-specific IGF-II is a major modulator of placental and fetal growth , 2002, Nature.

[27]  L. Raftery,et al.  TGFβ signaling at the summit , 2005 .

[28]  Janet Rossant,et al.  Gata 3 regulates trophoblast development downstream of Tead 4 and in parallel to Cdx 2 , 2022 .

[29]  R. Kellems,et al.  Transcription Factor AP-2γ Regulates Murine Adenosine Deaminase Gene Expression during Placental Development* , 1998, The Journal of Biological Chemistry.

[30]  J. Massagué,et al.  Mechanisms of TGF-beta signaling from cell membrane to the nucleus. , 2003, Cell.

[31]  A. Look,et al.  A Rap GTPase interactor, RADIL, mediates migration of neural crest precursors. , 2007, Genes & development.

[32]  Soumen Paul,et al.  GATA3 Is Selectively Expressed in the Trophectoderm of Peri-implantation Embryo and Directly Regulates Cdx2 Gene Expression* , 2009, The Journal of Biological Chemistry.

[33]  Y. Saijoh,et al.  Determination of left/right asymmetric expression of nodal by a left side-specific enhancer with sequence similarity to a lefty-2 enhancer. , 1999, Genes & development.

[34]  J. Smith,et al.  Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate , 1990, Nature.

[35]  H. Schorle,et al.  Transcription Factor Gene AP-2γ Essential for Early Murine Development , 2002, Molecular and Cellular Biology.

[36]  D. Norris,et al.  Cell fate decisions within the mouse organizer are governed by graded Nodal signals. , 2003, Genes & development.

[37]  H. Shibuya,et al.  TMEPAI, a transmembrane TGF-beta-inducible protein, sequesters Smad proteins from active participation in TGF-beta signaling. , 2010, Molecular cell.

[38]  P. Hoodless,et al.  Formation of the definitive endoderm in mouse is a Smad2-dependent process. , 2000, Development.

[39]  Jeffrey L. Wrana,et al.  TβRI Phosphorylation of Smad2 on Ser465 and Ser467 Is Required for Smad2-Smad4 Complex Formation and Signaling* , 1997, The Journal of Biological Chemistry.

[40]  A. Celeste,et al.  Nodal Signaling Uses Activin and Transforming Growth Factor-β Receptor-regulated Smads* , 2001, The Journal of Biological Chemistry.

[41]  Y. Saijoh,et al.  Left-right asymmetric expression of lefty2 and nodal is induced by a signaling pathway that includes the transcription factor FAST2. , 2000, Molecular cell.

[42]  J. Wrana,et al.  The MAD-Related Protein Smad7 Associates with the TGFβ Receptor and Functions as an Antagonist of TGFβ Signaling , 1997, Cell.

[43]  Michael M Shen,et al.  Two Modes by which Lefty Proteins Inhibit Nodal Signaling , 2004, Current Biology.

[44]  Ariel J. Levine,et al.  TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. , 2005, Development.

[45]  C. Zimmerman,et al.  Modulation of Smad2-mediated Signaling by Extracellular Signal-regulated Kinase* , 2002, The Journal of Biological Chemistry.

[46]  Konstantinos J. Mavrakis,et al.  Arkadia Enhances Nodal/TGF-β Signaling by Coupling Phospho-Smad2/3 Activity and Turnover , 2007, PLoS biology.

[47]  J. Massagué,et al.  A mechanism of repression of TGFbeta/ Smad signaling by oncogenic Ras. , 1999, Genes & development.

[48]  D. Norris,et al.  Asymmetric and node-specific nodal expression patterns are controlled by two distinct cis-acting regulatory elements. , 1999, Genes & development.

[49]  Y. Saijoh,et al.  Two-step regulation of left-right asymmetric expression of Pitx2: initiation by nodal signaling and maintenance by Nkx2. , 2001, Molecular cell.

[50]  Ana D. Lopez,et al.  Activin A Maintains Pluripotency of Human Embryonic Stem Cells in the Absence of Feeder Layers , 2005, Stem cells.

[51]  R. Mason,et al.  Inactivation of Smad-Transforming Growth Factor β Signaling by Ca2+-Calmodulin-Dependent Protein Kinase II , 2000, Molecular and Cellular Biology.

[52]  J. Graff,et al.  Smad3 Mutant Mice Develop Metastatic Colorectal Cancer , 1998, Cell.

[53]  Xuan Yuan,et al.  Activin A Maintains Self‐Renewal and Regulates Fibroblast Growth Factor, Wnt, and Bone Morphogenic Protein Pathways in Human Embryonic Stem Cells , 2006, Stem cells.

[54]  D Falb,et al.  The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. , 1997, Cell.

[55]  H. Schorle,et al.  Transcription factor gene AP-2 gamma essential for early murine development. , 2002, Molecular and cellular biology.

[56]  K. Miyazono,et al.  The N domain of Smad7 is essential for specific inhibition of transforming growth factor-β signaling , 2001, The Journal of cell biology.

[57]  E. Li,et al.  Smad2 role in mesoderm formation, left–right patterning and craniofacial development , 1998, Nature.

[58]  I. Katkov,et al.  Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells. , 2006, Cryobiology.

[59]  C. Heldin,et al.  Smad regulation in TGF-beta signal transduction. , 2001, Journal of cell science.

[60]  Joshua W. Vincentz,et al.  Fgf15 is required for proper morphogenesis of the mouse cardiac outflow tract , 2005, Genesis.

[61]  H. Aburatani,et al.  Chromatin Immunoprecipitation on Microarray Analysis of Smad2/3 Binding Sites Reveals Roles of ETS1 and TFAP2A in Transforming Growth Factor β Signaling , 2008, Molecular and Cellular Biology.

[62]  Konstantinos J. Mavrakis,et al.  Graded Smad2/3 Activation Is Converted Directly into Levels of Target Gene Expression in Embryonic Stem Cells , 2009, PloS one.

[63]  P. M. Nissom,et al.  A novel normalization method for effective removal of systematic variation in microarray data , 2006, Nucleic acids research.

[64]  J. Gurdon,et al.  Morphogen gradient interpretation , 2001, Nature.

[65]  K. Luo,et al.  Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein. , 1999, Science.

[66]  Janet Rossant,et al.  Interaction between Oct3/4 and Cdx2 Determines Trophectoderm Differentiation , 2005, Cell.

[67]  Hiroshi Hamada,et al.  Establishment of vertebrate left–right asymmetry , 2002, Nature Reviews Genetics.

[68]  I. Sandovici,et al.  Adaptation of nutrient supply to fetal demand in the mouse involves interaction between the Igf2 gene and placental transporter systems. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[69]  G. Martin,et al.  Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. , 1999, Genes & development.

[70]  G. Pan,et al.  NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. , 2008, Cell stem cell.

[71]  P. Lemaire,et al.  Activin signalling and response to a morphogen gradient , 1994, Nature.

[72]  Ariel J. Levine,et al.  TGFβ/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells , 2005 .

[73]  B. Herrmann,et al.  Expression pattern of the Brachyury gene in whole-mount TWis/TWis mutant embryos. , 1991, Development.

[74]  Alexander F. Schier,et al.  The zebrafish Nodal signal Squint functions as a morphogen , 2001, Nature.