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Directed differentiation of telencephalic precursors from embryonic stem cells

Nature Neuroscience volume 8, pages 288–296 (2005)Cite this article

Abstract

We demonstrate directed differentiation of telencephalic precursors from mouse embryonic stem (ES) cells using optimized serum-free suspension culture (SFEB culture). Treatment with Wnt and Nodal antagonists (Dkk1 and LeftyA) during the first 5 d of SFEB culture causes nearly selective neural differentiation in ES cells (∼90%). In the presence of Dkk1, with or without LeftyA, SFEB induces efficient generation (∼35%) of cells expressing telencephalic marker Bf1. Wnt3a treatment during the late culture period increases the pallial telencephalic population (Pax6+ cells yield up to 75% of Bf1+ cells), whereas Shh promotes basal telencephalic differentiation (into Nkx2.1+ and/or Islet1/2+ cells) at the cost of pallial telencephalic differentiation. Thus, in the absence of caudalizing signals, floating aggregates of ES cells generate naive telencephalic precursors that acquire subregional identities by responding to extracellular patterning signals.

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Figure 1: Efficient neural conversion of mouse ES cells in the SFEB culture with Dkk1 and LeftyA.
Figure 2: Selective biases do not have a major role in neural induction in SFEB.
Figure 3: Differentiation of Bf1+ cells in SFEB-treated ES cells and further enhancement by Dkk1 and LeftyA.
Figure 4: Differential control of dorsal and ventral telencephalic specification by soluble patterning signals.
Figure 5: Expression of ventral telencephalic markers by Wnt and Shh signals.

References

  1. Bain, G., Kitchens, D., Yao, M., Huettner, J.E. & Gottlieb, D.I. Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168, 342–357 (1995).

    Article  CAS  Google Scholar 

  2. Wichterle, H., Lieberam, I., Porter, J.A. & Jessell, T.M. Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397 (2002).

    Article  CAS  Google Scholar 

  3. Mizuseki, K. et al. Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc. Natl. Acad. Sci. USA 100, 5828–5833 (2003).

    Article  CAS  Google Scholar 

  4. Kawasaki, H. et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31–40 (2000).

    Article  CAS  Google Scholar 

  5. Lee, S.H., Lumelsky, N., Studer, L., Auerbach, J.M. & McKay, R.D. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat. Biotechnol. 18, 675–679 (2000).

    Article  CAS  Google Scholar 

  6. Ying, Q.L., Stavridis, M., Griffiths, D., Li, M. & Smith, A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21, 183–186 (2003).

    Article  CAS  Google Scholar 

  7. Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  Google Scholar 

  8. Martin, G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638 (1981).

    Article  CAS  Google Scholar 

  9. Aubert, J. et al. Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice. Proc. Natl. Acad. Sci. USA 100, 11836–11841 (2003).

    Article  CAS  Google Scholar 

  10. Wood, H.B. & Episkopou, V. Comparative expression of the mouse Sox1, Sox2 and Sox3 genes from pre-gastrulation to early somite stages. Mech. Dev. 86, 197–201 (1999).

    Article  CAS  Google Scholar 

  11. Haub, O. & Goldfarb, M. Expression of the fibroblast growth factor-5 gene in the mouse embryo. Development 112, 397–406 (1991).

    CAS  PubMed  Google Scholar 

  12. Hebert, J.M., Boyle, M. & Martin, G.R. mRNA localization studies suggest that murine FGF-5 plays a role in gastrulation. Development 112, 407–415 (1991).

    CAS  PubMed  Google Scholar 

  13. Aubert, J., Dunstan, H., Chambers, I. & Smith, A. Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. Nat. Biotechnol. 20, 1240–1245 (2002).

    Article  CAS  Google Scholar 

  14. Parisi, S. et al. Nodal-dependent Cripto signaling promotes cardiomyogenesis and redirects the neural fate of embryonic stem cells. J. Cell Biol. 163, 303–314 (2003).

    Article  CAS  Google Scholar 

  15. Finley, M.F., Devata, S. & Huettner, J.E. BMP-4 inhibits neural differentiation of murine embryonic stem cells. J. Neurobiol. 40, 271–287 (1999).

    Article  CAS  Google Scholar 

  16. Glinka, A. et al. Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391, 357–362 (1998).

    Article  CAS  Google Scholar 

  17. Sakuma, R. et al. Inhibition of Nodal signalling by Lefty mediated through interaction with common receptors and efficient diffusion. Genes Cells 7, 401–412 (2002).

    Article  CAS  Google Scholar 

  18. Pera, M.F. et al. Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J. Cell Sci. 117, 1269–1280 (2004).

    Article  CAS  Google Scholar 

  19. Doetschman, T.C., Eistetter, H., Katz, M., Schmidt, W. & Kemler, R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87, 27–45 (1985).

    CAS  PubMed  Google Scholar 

  20. Duncan, S.A., Nagy, A. & Chan, W. Murine gastrulation requires HNF-4 regulated gene expression in the visceral endoderm: tetraploid rescue of Hnf-4(−/−) embryos. Development 124, 279–287 (1997).

    CAS  PubMed  Google Scholar 

  21. Tao, W. & Lai, E. Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain. Neuron 8, 957–966 (1992).

    Article  CAS  Google Scholar 

  22. Xuan, S. et al. Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres. Neuron 14, 1141–1152 (1995).

    Article  CAS  Google Scholar 

  23. Oliver, G. et al. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121, 4045–4055 (1995).

    CAS  PubMed  Google Scholar 

  24. Nordstrom, U., Jessell, T.M. & Edlund, T. Progressive induction of caudal neural character by graded Wnt signaling. Nat. Neurosci. 5, 525–532 (2002).

    Article  Google Scholar 

  25. Piccolo, S. et al. The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature 397, 707–710 (1999).

    Article  CAS  Google Scholar 

  26. Ding, J. et al. Cripto is required for correct orientation of the anterior-posterior axis in the mouse embryo. Nature 395, 702–707 (1998).

    Article  CAS  Google Scholar 

  27. Puelles, L. et al. Pallial and subpallial derivatives in the embryonic chick and mouse telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and Tbr-1. J. Comp. Neurol. 424, 409–438 (2000).

    Article  CAS  Google Scholar 

  28. Hsieh-Li, H.M. et al. Gsh-2, a murine homeobox gene expressed in the developing brain. Mech. Dev. 50, 177–186 (1995).

    Article  CAS  Google Scholar 

  29. Lazzaro, D., Price, M., de Felice, M. & Di Lauro, R. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development 113, 1093–1104 (1991).

    CAS  PubMed  Google Scholar 

  30. Lee, S.M., Tole, S., Grove, E. & McMahon, A.P. A local Wnt-3a signal is required for development of the mammalian hippocampus. Development 127, 457–467 (2000).

    CAS  PubMed  Google Scholar 

  31. Galceran, J., Miyashita-Lin, E.M., Devaney, E., Rubenstein, J.L. & Grosschedl, R. Hippocampus development and generation of dentate gyrus granule cells is regulated by LEF1. Development 127, 469–482 (2000).

    CAS  PubMed  Google Scholar 

  32. Gunhaga, L. et al. Specification of dorsal telencephalic character by sequential Wnt and FGF signaling. Nat. Neurosci. 6, 701–707 (2003).

    Article  CAS  Google Scholar 

  33. Mukhopadhyay, M. et al. Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse. Dev. Cell 1, 423–434 (2001).

    Article  CAS  Google Scholar 

  34. Simeone, A., Acampora, D., Gulisano, M., Stornaiuolo, A. & Boncinelli, E. Nested expression domains of four homeobox genes in developing rostral brain. Nature 358, 687–690 (1992).

    Article  CAS  Google Scholar 

  35. Ericson, J. et al. Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube. Cell 81, 747–756 (1995).

    Article  CAS  Google Scholar 

  36. Chiang, C. et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407–413 (1996).

    Article  CAS  Google Scholar 

  37. Rallu, M. et al. Dorsoventral patterning is established in the telencephalon of mutants lacking both Gli3 and Hedgehog signaling. Development 129, 4963–4974 (2002).

    CAS  PubMed  Google Scholar 

  38. Wang, H.F. & Liu, F.C. Developmental restriction of the LIM homeodomain transcription factor Islet-1 expression to cholinergic neurons in the rat striatum. Neuroscience 103, 999–1016 (2001).

    Article  CAS  Google Scholar 

  39. Munoz-Sanjuan, I. & Brivanlou, A.H. Neural induction, the default model and embryonic stem cells. Nat. Rev. Neurosci. 3, 271–280 (2002).

    Article  CAS  Google Scholar 

  40. Tropepe, V. et al. Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 30, 65–78 (2001).

    Article  CAS  Google Scholar 

  41. Shirayoshi, Y., Okada, T.S. & Takeichi, M. The calcium-dependent cell-cell adhesion system regulates inner cell mass formation and cell surface polarization in early mouse development. Cell 35, 631–638 (1983).

    Article  CAS  Google Scholar 

  42. Kim, J.H. et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 418, 50–56 (2002).

    Article  CAS  Google Scholar 

  43. Barberi, T. et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat. Biotechnol. 21, 1200–1207 (2003).

    Article  CAS  Google Scholar 

  44. Nieuwkoop, P.D. Activation and organization of the central nervous system in amphibians. J. Exp. Zool. 120, 1–81 (1952).

    Article  Google Scholar 

  45. Takahashi, K., Liu, F.C., Hirokawa, K. & Takahashi, H. Expression of Foxp2, a gene involved in speech and language, in the developing and adult striatum. J. Neurosci. Res. 73, 61–72 (2003).

    Article  CAS  Google Scholar 

  46. Chang, C.W. et al. Identification of a developmentally regulated striatum-enriched zinc-finger gene, Nolz-1, in the mammalian brain. Proc. Natl. Acad. Sci. USA 101, 2613–2618 (2004).

    Article  CAS  Google Scholar 

  47. Hirashima, M. et al. A chemically defined culture of VEGFR2+ cells derived from embryonic stem cells shows the role of VEGFR1 in tuning the threshold for VEGF in developing endothelial cells. Blood 101, 2261–2267 (2003).

    Article  CAS  Google Scholar 

  48. Johansson, B.M. & Wiles, M.V. Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development. Mol. Cell. Biol. 15, 141–151 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to members of the Sasai lab and to T. Era, S. Kuratani, R. Ladher, I. Matsuo, F. Matsuzaki, H. Niwa, T. Watabe, R. Shigemoto and S. Kaneko for invaluable comments and advice on this work; to S. Nishikawa, S.-i. Nishikawa and M. Takeichi for labeled E-cadherin antibody; to A. Smith for Sox1-GFP ES cells and to N. Sasai for advice on antibody production. K.W. is thankful to S. Iwamizu-Watanabe for discussion and constant encouragement. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (Y.S., Y.W.), the Kobe Cluster Project (Y.S.) and the Leading Project (Y.S.).

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Authors and Affiliations

  1. Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe, 650-0047, Japan

    Kiichi Watanabe, Daisuke Kamiya, Ayaka Nishiyama, Tomoko Katayama, Kenji Mizuseki & Yoshiki Sasai

  2. Department of Medical Embryology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan

    Kiichi Watanabe, Daisuke Kamiya & Yoshiki Sasai

  3. Department of Physiology, Graduate School of Medicine, Osaka City University, Osaka, 545-8585, Japan

    Satoshi Nozaki & Yasuyoshi Watanabe

  4. Department of Molecular and System Neurobiology, Graduate School of Medicine, University of Tokyo, Tokyo, 113-0033, Japan

    Hiroshi Kawasaki

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Supplementary information

Supplementary Fig. 1

Immunostaining analysis of SFEB-treated ES cell aggregates. (PDF 1190 kb)

Supplementary Fig. 2

Analysis of the effects of Wnt, Nodal and BMP antagonists. (PDF 650 kb)

Supplementary Fig. 3

Expression of primitive endodermal and mesodermal markers in SFEB. (PDF 1019 kb)

Supplementary Fig. 4

BrdU uptake and TUNEL analyses in SFEB/BMP-treated ES cells. (PDF 207 kb)

Supplementary Fig. 5

Expression of rostral-caudal markers in differentiating ES cells. (PDF 998 kb)

Supplementary Fig. 6

RT-PCR analysis and the role of E-cad function in SDIA-treated ES cells. (PDF 1086 kb)

Supplementary Fig. 7

Systematic induction of pallial and subpallial telencephalic tissues. (PDF 486 kb)

Supplementary Fig. 8

Expression of basal telencephalic markers in SFEB/Shh-induced cells and the embryonal brain. (PDF 1246 kb)

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Watanabe, K., Kamiya, D., Nishiyama, A. et al. Directed differentiation of telencephalic precursors from embryonic stem cells. Nat Neurosci 8, 288–296 (2005). https://doi.org/10.1038/nn1402

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