LacZ Staining Cells fixed with 2% formaldehyde were stained for b-Gal using standard procedures. Supporting Information Table S1 Cell-context specific Notch1 target genes. List of cell- context specific Notch1 target genes identified by genome wide expression arrays in ESC under ectodermal and mesodermal differentiation conditions and in mesodermal progenitor cells. Found at: doi:10.1371/journal.pone.0011481.s001 (0.37 MB XLS) Table S2 Gene expression assays of Notch1 target genes. Summary of gene expression assays used to determine relative expression levels of 30 potential Notch1 target genes after induction of Notch signaling. Found at: doi:10.1371/journal.pone.0011481.s002 (0.02 MB XLS) References 1. Artavanis-Tsakonas S, Rand MD, Lake RJ (1999) Notch signaling: cell fate control and signal integration in development. Science 284: 770–776. 2. Lai EC (2004) Notch signaling: control of cell communication and cell fate. Development 131: 965–973. 3. Kopan R, Cagan R (1997) Notch on the cutting edge. Trends Genet 13: 465–467. 4. Hurlbut GD, Kankel MW, Lake RJ, Artavanis-Tsakonas S (2007) Crossing path with Notch in the hyper-network. Curr Opin Cell Biol 19: 166–175. 5. Iso T, Kedes L, Hamamori Y (2003) HES and HERP families: Multiple effectors of the Notch signaling pathway. J Cell Physiol 194: 237–255. 6. Leimeister C, Externbrink A, Klamt B, Gessler M (1999) Hey genes: A novel subfamily of hairy- and enhancer of split related genes specifically expressed during mouse embryogenesis. Mech Dev 85: 173–177. 7. Krejci A, Bernard F, Housden BE, Collins S, Bray SJ (2009) Direct response to Notch activation: Signaling crosstalk and incoherent logic. Sci Signal 2: 1–14. 8. Poellinger L, Lendahl U (2008) Modulating Notch signaling by pathway-intrinsic and pathway-extrinsic mechanisms. Curr Opin Genet Dev 18: 449–454. 9. Cormier S, Vandormael-Pournin S, Babinet C, Cohen-Tannoudji M (2004) Developmental expression of the Notch signaling pathway genes during mouse preimplantation development. Gene Expr Patterns 4: 713–717. 10. Nemir M, Croquelois A, Pedrazzini T, Radtke F (2006) Induction of cardiogenesis in embryonic stem cells via downregulation of Notch1 signaling. Circ Res 98: 1471–1478. 11. Swiatek PJ, Lindsell CE, del Amo FF, Weinmaster G, Gridley T (1994) Notch1 is essential for postimplantation development in mice. Genes & Dev 8: 707–719. 12. Oka C, Nakano T, Wakeham A, de la Pompa JL, Mori C, et al. (1995) Disruption of the mouse RBP-J kappa gene results in early embryonic death. Development 121: 3291–3301. 13. Conlon RA, Reaume AG, Rossant J (1995) Notch1 is required for the coordinate segmentation of somites. Development 121: 1533–1545. 14. Schroeder T, Meier-Stiegen F, Schwanbeck R, Eilken H, Nishikawa S, et al. (2006) Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development. Mech Dev 123: 570–579. 15. Lowell S, Benchoua A, Heavey B, Smith A (2006) Notch promotes neural lineage entry by pluripotent embryonic stem cells. PLoS Biology 4: e121. 16. Schroeder T, Kohlhof H, Rieber N, Just U (2003) Notch signaling induces multilineage myeloid differentiation and up-regulates PU.1 expression. J Immunol 170: 5538–5548. 17. Schwanbeck R, Schroeder T, Henning K, Kohlhof H, Rieber N, et al. (2008) Notch signaling in embryonic and adult myelopoiesis. Cells Tissues Organs 188: 91–102. 18. Henning K, Schroeder T, Schwanbeck R, Rieber N, Bresnick EH, et al. (2007) mNotch1 signaling and erythropoietin cooperate in erythroid differentiation of multipotent progenitor cells and upregulate beta-globin. Exp Hematol 35: 1321–1332. 19. Main H, Lee KL, Yang H, Haapa-Paananen S, Edgren H, et al. (2010) Interactions between Notch- and hypoxia-induced transcriptomes in embryonic stem cells. Exp Cell Res 316: 1610–24. 20. Taylor MK, Yeager K, Morrison SJ (2007) Physiological Notch signaling promotes gliogenesis in the developing perpheral and central nervous system. Development 134: 2435–2447. 21. Muto A, Iida A, Satoh S, Watanabe S (2009) The group E Sox genes Sox8 and Sox9 are regulated by Notch signaling and are required for Mu¨ller glial cell development in mouse retina. Exp Eye Res 89: 549–558. Context-Specific Notch Targets Video S1 PCA shows that samples of each cell population, i.e. ESC (ESCe and ESCm) and mesodermal progenitor cells (Mesoderm), cluster together, regardless of the expression of the NERT protein or the presence of OHT. Found at: doi:10.1371/journal.pone.0011481.s003 (5.51 MB MOV) Acknowledgments We thank Thomas Braun for providing the Myf5-LacZ-tk knock-in ESC, Shin-Ichi Nishikawa for providing the Runx1-Venus knock-in ESC, Michael Cross for critical reading of the manuscript and Silke Horn for expert technical assistance. Author Contributions Conceived and designed the experiments: UJ. Performed the experiments: FMS RS KB SM TH. Analyzed the data: FMS RS KB SM TH DR MZ UJ. Wrote the paper: RS UJ. 22. Zong Y, Panikkar A, Xu JF, Antoniou A, Raynaud P, et al. (2009) Notch signaling controls liver development by regulating biliary differentiation. Development 136: 1727–1739. 23. Osumi N, Shinohara H, Numayama-Tsuruta K, Maekawa M (2008) Pax6 transcription factor contributes to both embryonic and adult neurogenesis as a multifunctional regulator. Stem Cells 26: 1663–1672. 24. Suter DM, Tirefort D, Julien S, Krause KH (2009) A Sox1 to Pax6 switch drives neuroectoderm to radial glia progression during differentiation of mouse embryonic stem cells. Stem Cells 27: 49–58. 25. Onuma Y, Takahashi S, Asashima M, Kurata S, Gehring WJ (2002) Conservation of Pax 6 function and upstream activation by Notch signaling in eye development of frogs and flies. Proc Natl Acad Sci U S A 99: 2020–2025. 26. Levanon D, Groner Y (2004) Structure and regulated expression of mammalian RUNX genes. Oncogene 23: 4211–4219. 27. Kurokawa M (2006) AML1/Runx1 as a versatile regulator of hematopoiesis: Regulation of its function and a role in adult hematopoiesis. Int J Hematol 84: 136–142. 28. Lebestky T, Jung SH, Banerjee U (2003) A Serrate-expressing signaling center controls Drosophila hematopoiesis. Genes & Dev 17: 348–353. 29. Nakagawa M, Ichikawa M, Kumano K, Goyama S, Kawazu M, et al. (2006) AML1/Runx1 rescues Notch1-null mutation-induced deficiency of para-aortic splanchnopleural hematopoiesis. Blood 108: 3329–3334. 30. Burns CE, Traver D, Mayhall E, Shepard JL, Zon LI (2005) Hematopoietic stem cell fate is established by the Notch-Runx pathway. Genes & Dev 19: 2331–2342. 31. Vasyutina E, Lenhard D, Birchmeier C (2007) Notch function in myogenesis. Cell Cycle 6: 1451–1454. 32. Arnold HH, Braun T (2000) Genetics of muscle determination and development. Curr Top Dev Biol 48: 129–164. 33. Vasyutina E, Lenhard D, Wende H, Erdmann E, Epstein J, et al. (2007) RBP-J (Rbpsuh) is essential to maintain muscle progenitor cells and to generate satellite cells. Proc Natl Acad Sci U S A 104: 4443–4448. 34. Schuster-Gossler K, Cordes R, Gossler A (2007) Premature myogenic differentiation and depletion of progenitor cells cause severe muscle hypotrophy in Delta1 mutants. Proc Natl Acad Sci U S A 104: 537–542. 35. Kuroda K, Tani S, Tamura K, Minoguchi S, Kurooka H, et al. (1999) Delta- induced Notch signaling mediated by RBP-J inhibits MyoD expression and myogenesis. J Biol Chem 274: 7238–7244. 36. Asakura A, Hirai H, Kablar B, Morita S, Ishibashi J, et al. (2007) Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle. Proc Natl Acad Sci USA 104: 16552–16557. 37. Gensch N, Borchardt T, Schneider A, Riethmacher D, Braun T (2008) Different autonomous myogenic cell populations revealed by ablation of Myf5-expressing cells during mouse embryogenesis. Development 135: 1597–1604. 38. Haldar M, Karan G, Tvrdik P, Capecchi MR (2008) Two cell lineages, myf5 and myf5-independent, participate in mouse skeletal myogenesis. Dev Cell 14: 437–445. 39. Ustanina S, Carvajal J, Rigby P, Braun T (2007) The myogenic factor Myf5 supports efficient skeletal muscle regeneration by enabling transient myoblast amplification. Stem Cells 25: 2006–2016. 40. Buas M, Kabak S, Kadesh T (2009) Inhibition of myogenesis by Notch: Evidence for multiple pathways. J Cell Physiol 218: 84–93. 41. Ruzinova MB, Benezra R (2003) Id proteins in development, cell cycle and cancer. Trends Cell Biol 13: 410–418. 42. Baonza A, de Celis JF, Garcia-Bellido A (2000) Relationships between extramacrochaetae and Notch signalling in Drosophila wing development. Development 127: 2383–2393. PLoS ONE | www.plosone.org 17 July 2010 | Volume 5 | Issue 7 | e11481