Abstract
We portrayed the Notch system in embryonic stem cell (ESC)-derived embryoid bodies (EBs) differentiating under the standard protocols used to assess yolk sac (YS) hematopoiesis in vitro. Notch receptors and Notch ligands were detected in virtually all cells throughout EB development. Notch1 and Notch 2, but not Notch4, were visualized in the nucleus of EB cells, and all these receptors were also observed as patent cytoplasmic foci. Notch ligands (Delta-like1 and 4, Jagged1 and 2) were immunodetected mostly as cytoplasmic foci. Widespread Notch1 activation was evident at days 2–4 of EB differentiation, the time window of hemangioblast generation in this in vitro system. EBs experienced major spatial remodeling beyond culture day 4, the time point coincident with the transition between primitive and multilineage waves of YS hematopoiesis in vitro. At day 6, where definitive YS hematopoiesis is established in EBs, these exhibit an immature densely packed cellular region (DCR) surrounded by a territory of mesodermal-like cells and an outer layer of endodermal cells. Immunolabeling of Notch receptors and ligands was usually higher in the DCR. Our results show that Notch system components are continuously and abundantly expressed in the multicellular environments arising in differentiating EBs. In such an active Notch system, receptors and ligands do not accumulate extensively at the cell surface but instead localize at cytoplasmic foci, an observation that fits current knowledge on endocytic modulation of Notch signaling. Our data thus suggest that Notch may function as a territorial modulator during early development, where it may eventually influence YS hematopoiesis.
References
1.
Baldwin, R.J., G.B. Ten Dam, T.H. van Kuppevelt, G. Lacaud, J.T. Gallagher, V. Kouskoff, C.L. Merry (2008) A developmentally regulated heparan sulfate epitope defines a subpopulation with increased blood potential during mesodermal differentiation. Stem Cells 26: 3108–3118.
2.
Baron, M.H. (2005) Developmental Hematopoiesis: Methods and Protocols. Totowa, Humana Press, p 105.
3.
Berge, D., W. Koole, C. Fuerer, M. Fish, E. Eroglu, R. Nusse (2008) Wnt signaling mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell 3: 508–518.
4.
Botquin, V., H. Hess, G. Fuhrmann, C. Anastassiadis, M.K. Gross, G. Vriend, H.R. Schoeler (1998) New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2. Genes Dev 12: 2073–2090.
5.
Bray, S.J. (2006) Notch signaling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7: 678–689.
6.
Cheng, X., T.L. Huber, V.C. Chen, P. Gadue, G.M. Keller (2008) Numb mediates the interaction between Wnt and Notch to modulate primitive erythropoietic specification from the hemangioblast. Development 135: 3447–3458.
7.
Chiba, S. (2006) Notch Signaling in Stem Cell Systems. Stem Cells 24: 2437–2447.
8.
Cidadão, A.J., J.F. David-Ferreira (1986) A method for TEM visualization of the extracellular matrix three-dimensional organization in tissues. J Microsc 142: 49–62.
9.
Croce, J.C., D.R. McClay (2010) Dynamics of Delta/Notch signaling on endomesoderm segregation in the sea urchin embryo. Development 137: 83–91.
10.
Cumano, A., I. Godin (2007) Ontogeny of the hematopoietic system. Annu Rev Immunol 25: 745–785.
11.
Daley, G.Q. (2003) From embryos to embryoid bodies. Generating blood from embryonic stem cells. Ann NY Acad Sci 996: 122–131.
12.
Das, A.V., J. James, S. Bhattacharya, A.N. Imbalzano, M.L. Antony, G. Hedge, X. Zhao, K. Mallya, F. Ahmad, E. Knudsen, I. Ahmad (2007) SWI/SNF chromatin remodeling ATPase Brm regulates the differentiation of early retinal stem cells progenitors by influencing Brn3b expression and Notch signaling. J Biol Chem 282: 35187–35201.
13.
Del Monte, G., J. Grego-Bessa, A. González-Rajal, V. Bolós, J.L. De La Pompa (2007) Monitoring Notch1 activity in development: evidence for a feedback regulatory loop. Dev Dyn 236: 2594–2614.
14.
Fiúza, U.M., A.M. Arias (2007) Cell and molecular biology of Notch. J Endocrinol 194: 459–474.
15.
Ge, C., T. Liu, X. Hou, P. Stanley (2008) In vivo consequences of deleting EGF repeats 8–12 including the ligand binding domain of mouse Notch1. BMC Dev Biol 8: 48.
16.
Gering, M., R. Patient (2010) Notch signalling and haematopoietic stem cell formation during embryogenesis. J Cell Physiol 222: 11–16.
17.
Hadland, B.K., S.S. Huppert, J. Kanungo, Y. Xue, R. Jiang, T. Gridley, R.A. Conlon, A.M. Cheng, R. Kopan, G.D. Longmore (2004) A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis. Blood 104: 3097–3105.
18.
Hayashi, Y., M.K. Furue, T. Okamoto, K. Ohnuma, Y. Myoishi, Y. Fukuhara, T. Abe, J.D. Sato, R. Hata, M. Asashima (2007) Integrins regulate mouse embryonic stem cell self-renewal. Stem Cells 25: 3005–3015.
19.
Hayward, P., T. Kalmar, A. Martinez, A. Arias (2008) Wnt/Notch signaling and information processing during development. Development 135: 411–424.
20.
Hurlbut, G., M.W. Kankel, R.J. Lake, S. Artavanis-Tsakonas (2007) Crossing paths with Notch in the hyper-network. Curr Opin Cell Biol 19: 1–10.
21.
Ito, Y., M. Hikino, Y. Yajima, T. Mikami, S. Sirko, A. von Holst, A. Faissner, S. Fukui, K. Sugahara (2005) Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56, and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library. Glycobiology 15: 593–603.
22.
Jaleco, A.C., H. Neves, E. Hooijberg, P. Gameiro, N. Clode, M. Haury, D. Henrique, L. Parreira (2001) Differential effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid differentiation. J Exp Med 194: 991–1002.
23.
Kadam, S., B.M. Emerson (2003) Transcriptional specificity of human SWI/SNF BRG1 and BRM chromatin remodeling complexes. Moll Cell 11: 377–389.
24.
Keller, G. (2005) Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev 19: 1129–1155.
25.
Klosen, P., X. Maessen, B.P. Aguilar (1993) PEG embedding for immuno-cytochemistry: application to the analysis of immunoreactivity loss during histological processing. J Histochem Cytochem 41: 455–463.
26.
Kobayashi, T., R. Kageyama (2010) Hes1 regulates embryonic stem cell differentiation by suppressing Notch signaling. Genes Cells 15: 689–698.
27.
Kopan, R., M.X.G. Ilagan (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137: 216–232.
28.
Kyba, M., R.C. Perlingeiro, G.Q. Daley (2003) Development of hematopoietic repopulating cells from embryonic stem cells. Methods Enzymol 365: 114–129.
29.
Lanner, F., K.L. Lee, M. Sohl, K. Holmborn, H. Yang, J. Wilbertz, L. Poellinger, J. Rossant, F. Farnebo (2010) Heparan sulfation-dependent fibroblast growth factor signaling maintains embryonic stem cells primed for differentiation in a heterogeneous state. Stem Cells 28: 191–200.
30.
Li, X., P. Leder (2007) Identifying genes preferentially expressed in undifferentiated embryonic stem cells. BMC Cell Biol 8: 37.
31.
Ling, V., S. Neben (1997) In vitro differentiation of embryonic stem cells: immunophenotypic analysis of cultured embryoid bodies. J Cell Physiol 171: 104–115.
32.
Mowery, J., J. Chesner, S. Spangenberger, D.C. Hixson (1989) Rapid low molecular weight polyethylene glycol embedding protocol for immunocytochemistry. J Histochem Cytochem 37: 1549–1552.
33.
Murry, C.E., G. Keller (2008) Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132: 661–680.
34.
Nairn, A.V., A. Kinoshita-Toyoda, H. Toyoda, J. Xie, K. Harris, S. Dalton, M. Kulik, J.M. Pierce, T. Toida, K.W. Moremen, R.J. Linhardt (2007) Glycomics of proteoglycan biosynthesis in murine embryonic stem cell differentiation. J Proteome Res 6: 4374–4387.
35.
Neves, H., F. Weerkamp, A.C. Gomes, B.A. Naber, P. Gameiro, J.D. Becker, P. Lúcio, N. Clode, J.J. van Dongen, F.J. Staal, L. Parreira (2006) Effects of Delta1 and Jagged1 on early human hematopoiesis: correlation with expression of Notch signaling-related genes in CD34+ cells. Stem Cells 24: 1328–1337.
36.
Noggle, S.A., D. Weiler, B.G. Condie (2006) Notch signaling is inactive but inducible in human embryonic stem cells. Stem Cells 24:1646–1653.
37.
Orkin, S.H., L.I. Zon (2008) Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132: 631–644.
38.
Ramasamy, S.K., N. Lenka (2010) Notch exhibits ligand bias and manoeuvres stage specific steering of neural differentiation in ESCs. Mol Cell Biol 30: 1946–1957.
39.
Revinski, D.R., A.R. Paganelli, A.E. Carrasco, S.L. López (2010) Delta-Notch signaling is involved in the segregation of the three germ layers in Xenopus laevis. Dev Biol 339: 477–492.
40.
Sasaki, N., T. Sasamura, H.O. Ishikawa, M. Kanai, R. Ueda, K. Saigo, K. Matsuno (2007) Polarized exocytosis and transcytosis of Notch during its apical localization in Drosophila epithelial cells. Genes Cells 12: 89–103.
41.
Schiedlmeier, B., A.C. Santos, A. Ribeiro, N. Moncaut, D. Lesinski, H. Auer, K. Kornacker, W. Ostertag, C. Baum, M. Mallo, H. Klump (2007) HOXB4’s road map to stem cell expansion. Proc Natl Acad Sci USA 104: 16952–16957.
42.
Shi, S., P. Stanley (2006) Evolutionary origins of Notch signaling in early development. Cell Cycle 5: 274–278.
43.
Sorensen, E.B., S.D. Conner (2010) Gamma secretase-dependent cleavage initiates Notch signaling from the plasma membrane. Traffic 11: 1234–1245.
44.
Stahl, M., K. Uemura, C. Ge, S. Shi, Y. Tashima, P. Stanley (2008) Roles of Pofut1 and O-fucose in mammalian Notch signaling. J Biol Chem 283: 13638–13651.
45.
Stier, S., Y. Ko, R. Forkert, C. Lutz, T. Neuhaus, E. Grünewald, T. Cheng, D. Dombkowski, L.M. Calvi, S.R. Rittling, D.T. Scadden (2005) Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. J Exp Med 201: 1781–1791.
46.
Takeuchi, J.K., H. Lickert, B.W. Bisgrove, X. Sun, M. Yamamoto, K. Chawengsaksophak, H. Hamada, H.J. Yost, J. Rossant, B.G. Bruneau (2007) Baf60c is a nuclear Notch signaling component required for the establishment of left-right asymmetry. Proc Natl Acad Sci USA 104: 846–851.
47.
Tanaka, M., M. Kokubo, T. Marunouchi (2007) Asymmetric localization of Notch2 on the microvillous surface of choroid plexus epithelial cells. Histochem Cell Biol 127: 449–456.
48.
Wolosewick, J.J. (1980) The application of polyethylene glycol (PEG) to electron microscopy. J Cell Biol 86: 661–675.
49.
Yu, X., J. Zou, Z. Ye, H. Hammond, G. Chen, A. Tokunaga, P. Mali, Y.M. Li, C. Civin, N. Gaiano, L. Cheng (2008) Notch signaling activation in human embryonic stem cells is required for embryonic, but not trophoblastic, lineage commitment. Cell Stem Cell 2: 461–471.
50.
Zagouras, P., S. Stifani, C.M. Blaumueller, M.L. Carcangiu, S. Artavanis-Tsakonas (1995) Alterations of Notch signaling in neoplastic lesions of the human cervix. Proc Natl Acad Sci USA 92: 6414–6418.
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2010
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