Culture of Embryonic Cells for Analysis of Amphibian and Mammalian Early Embryogenesis

  • Norio Nakatsuji
  • Koichiro Hashimoto
Part of the Bodega Marine Laboratory Marine Science Series book series (BMSS)


Culture of embryonic cells isolated from early embryos allows detailed analysis of the cell motility, interactions between cells, and between cells and the extracellular matrix (ECM). There is always the risk that cell behavior in culture might be artifacts not related to the behavior inside embryos. In many situations, one must use cells immediately after isolation from embryos, and try to prepare culture conditions similar to those inside the embryo.

Migration of the presumptive mesodermal cells during amphibian gastrulation was studied by such methods. It was first necessary to find culture conditions to allow mesodermal cells to behave in a manner similar to that found inside embryos. By the use of culture medium with a pH and calcium ion concentration found in embryos, as well as coating of the substratum with extracellular matrix (ECM) components, we were able to perform detailed analysis of cell behavior. Our analysis revealed an important role for the ECM fibril network containing fibronectin as a substratum which guided mesodermal cell migration by contact guidance.

A similar strategy was used for the analysis of mammalian gastrulation. Mesodermal cells isolated from the primitive-streak-stage mouse embryos attached to and migrated on the ECM produced by endothelial cells. This culture system revealed a deficiency in the mesodermal cells isolated from Brachyury (T) mutant embryos. Cell attachment to ECM was further analyzed by using antibodies against fibronectin or laminin and synthetic peptides, or by coating of the culture substratum with various ECM components. These studies indicated that both fibronectin and laminin play roles in adhesion and migration of the mesodermal cells.

For longer range analysis, however, it would be advantageous if isolated embryonic cells could be re-introduced into embryos after manipulations in culture. Genetical manipulation of the isolated cells would produce insight into the molecular basis of embryogenesis, but it requires a long term culture of the embryonic cells without losing the ability to re-integrate into the embryo. Mouse embryonic stem (ES) cells have such characteristics. We introduced a marker gene (Lac Z) into ES cells and analyzed early embryogenesis by producing chimaeric mouse embryos.


Mouse Embryo Embryonic Cell Inner Cell Mass Primitive Streak Mesodermal Cell 
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  1. Boucaut, J.-C, T. Darribère, D. Shi, J.-F. Riou, K.E. Johnson, and M. Delarue. 1991. Amphibian Gastrulation: The Molecular Bases of Mesodermal Cell Migration in Urodel Embryos, p. 169–184. In: Gastrulation: Movements, Patterns, and Molecules R. Keller, W.H. Clark Jr., F. Griffin (Eds.). Plenum Press, New York.Google Scholar
  2. Boucaut, J.-C. and T. Darribère. 1983a. Fibronectin in early amphibian embryos: Migrating mesodermal cells are in contact with a fibronectin-rich fibrillar matrix established prior to gastrulation. Cell Tissue Res. 234:135–145.PubMedCrossRefGoogle Scholar
  3. Boucaut, J.-C. and T. Darribère. 1983b. Presence of fibronectin during early embryogenesis in the amphibian Pleurodeles waltl Cell Differ. 12:77–83.CrossRefGoogle Scholar
  4. Darribère, T., H. Boulekbache, D.L. Shi, and J.-C. Boucaut. 1985. Immunoelectron microscopic study of fibronectin in gastrulating amphibian embryos. Cell Tissue Res. 239:75–80.CrossRefGoogle Scholar
  5. Darribère, T., J.-F. Riou, D.L. Shi, M. Delarue, and J.-C. Boucaut. 1986. Synthesis and distribution of laminin related polypeptides in early amphibian embryos. Cell Tissue Res. 246:45–51.PubMedCrossRefGoogle Scholar
  6. Dufour, S., J.-L. Duband, A.R. Kornblihtt, and J.P. Thiery. 1988. The role of fibronectins in embryonic cell migration. Trends Genet. 4:198–203.PubMedCrossRefGoogle Scholar
  7. Hashimoto, K, H. Fujimoto, and N. Nakatsuji. 1987. An ECM substratum allows mouse mesodermal cells isolated from the primitive streak to exhibit motility similar to that inside embryo, and it reveals a deficiency in the T/T mutant cells. Development 100:587–598.PubMedGoogle Scholar
  8. Hashimoto, K. and N. Nakatsuji. 1989. Formation of the primitive streak and mesoderm cells in mouse embryos—detailed scanning electron microscopical study. Dev. Growth & Differ. 31:209–218.CrossRefGoogle Scholar
  9. Heasman, J., C.C. Wylie, P. Hausen, and J.C. Smith. 1984. Fates and states of determination of single vegetal pole blastomeres of Xenopus laevis. Cell 37:185–194.CrossRefGoogle Scholar
  10. Herrmann, B.G., S. Labeit, A. Poustka, T.R. King, and H. Lehrach. 1990. Cloning of the T gene required in mesoderm formation in the mouse. Nature 343:617–622.PubMedCrossRefGoogle Scholar
  11. Humphries, M.J., S.K. Akiyama, A. Komoriya, K. Olden, and KM. Yamada. 1986. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. J. Cell Biol. 103:2637–2647.PubMedCrossRefGoogle Scholar
  12. Humphries, M.J., S.K. Akiyama, A. Komoriya, K. Olden, and KM. Yamada. 1988. Neurite extension of chicken peripheral nervous system neurons on fibronectin: Relative importance of specific adhesion sites in the central cell-binding domain and the alternatively spliced type III connecting segment. J. Cell Biol. 106:1289–1297.PubMedCrossRefGoogle Scholar
  13. Humphries, M.J., A. Komoriya, S.K. Akiyama, K. Olden, and KM. Yamada. 1987. Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion. J. Biol. Chem. 262: 6886–6892.PubMedGoogle Scholar
  14. Johnson, K.E., N. Nakatsuji, and J.-C. Boucaut. 1990. Extracellular matrix control of cell migration during amphibian gastrulation. p. 349–374. In: Cytoplasmic Organization Systems: Primers in Developmental Biology. G.M. Malacinski (Ed.). McGraw-Hill, New York.Google Scholar
  15. Kadokawa, Y., Y. Kato, and G. Eguchi. 1987. Cell lineage analysis of the primitive and visceral endoderm of mouse embryos cultured in vitro. Cell Differ. 21:69–76.Google Scholar
  16. Kadokawa, Y., H. Suemori, and N. Nakatsuji. 1990. Cell lineage analyses of epithelia and blood vessels in chimeric mouse embryos by use of an embryonic stem cell line expressing the β-galactosidase gene. Cell Differ. Dev. 29:187–194.PubMedCrossRefGoogle Scholar
  17. Lawson, K.A., J.J. Meneses, and R.A. Pedersen. 1986. Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer. Dev. Biol. 115:325–339.PubMedCrossRefGoogle Scholar
  18. Martin, G.R. 1981. 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.PubMedCrossRefGoogle Scholar
  19. Nakatsuji, N. 1975. Studies on the gastrulation of amphibian embryos: Cell movement during gastrulation in Xenopus laevis embryos. Wilhelm Roux’s Arch. Dev. Biol. 178:1–14.CrossRefGoogle Scholar
  20. Nakatsuji, N. 1984. Cell locomotion and contact guidance in amphibian gastrulation. Am. Zool. 24:615–627.Google Scholar
  21. Nakatsuji, N. 1986. Presumptive mesodermal cells from Xenopus laevis gastrulae attach to and migrate on substrata coated with fibronectin or laminin. J. Cell Sci. 86:109–118.PubMedGoogle Scholar
  22. Nakatsuji, N., A. Gould, and K.E. Johnson. 1982. Movement and guidance of migrating mesodermal cells in Ambystoma maculatum gastrulae. J. Cell Sci. 56:207–222.PubMedGoogle Scholar
  23. Nakatsuji, N., K. Hashimoto, and M. Hayashi. 1985. Laminin fibrils in newt gastrulae visualized by immunofluorescent staining. Dev. Growth & Differ. 27:639–643.CrossRefGoogle Scholar
  24. Nakatsuji, N. and K.E. Johnson. 1982. Cell locomotion in vitro by Xenopus laevis gastrula mesodermal cells. Cell Motil. 2:149–161.PubMedGoogle Scholar
  25. Nakatsuji, N. and K.E. Johnson. 1983a. Comparative study of extracellular fibrils on the ectodermal layer in gastrulae of five amphibian species. J. Cell Sci. 59:61–70.PubMedGoogle Scholar
  26. Nakatsuji, N. and K.E. Johnson. 1983b. Conditioning of a culture substratum by the ectodermal layer promotes attachment and oriented locomotion by amphibian gastrula mesodermal cells. J. Cell Sci. 59:43–60.PubMedGoogle Scholar
  27. Nakatsuji, N. and K.E. Johnson. 1984. Experimental manipulation of a contact guidance system in amphibian gastrulation by mechanical tension. Nature 307:453–455.PubMedCrossRefGoogle Scholar
  28. Nakatsuji, N., M.A. Smolira, and C.C. Wylie. 1985. Fibronectin visualized by scanning electron microscopy immunocytochemistry on the substratum for cell migration in Xenopus laevis gastrulae. Dev. Biol. 107:264–268.PubMedCrossRefGoogle Scholar
  29. Nakatsuji, N., M.H.L. Snow, and C.C. Wylie. 1986. Cinemicrographic study of the cell movement in the primitive-streak-stage mouse embryo. J. Embryol. Exp. Morphol. 96:99–109.PubMedGoogle Scholar
  30. Robertson, E.J. 1987. Embryo-derived stem cell lines, p. 71–112. In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E.J. Robertson (Ed.). IRL Press, Oxford.Google Scholar
  31. Ruoslahti, E. and M.D. Pierschbacher. 1987. New perspectives in cell adhesion: RGD and integrins. Science 238:491–497.PubMedCrossRefGoogle Scholar
  32. Shi, D.-L., T. Darribère, K.E. Johnson, and J.-C. Boucaut. 1989. Initiation of mesodermal cell migration and spreading relative to gastrulation in the urodele amphibian Pleurodeles waltl. Development 105:351–363.Google Scholar
  33. Snow, M.H.L. 1978. Techniques for separating early embryonic tissues, p. 167–178. In: Methods in Mammalian Reproduction. J.C. Daniel, Jr. (Ed.). Academic Press, New York.Google Scholar
  34. Suemori, H., Y. Kadokawa, K. Goto, I. Araki, H. Kondoh, and N. Nakatsuji. 1990. A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous β-galactosidase expression. Cell Differ. Dev. 29:181–186.PubMedCrossRefGoogle Scholar
  35. Yanagisawa, K.O., H. Fujimoto, and H. Urushibara. 1981. Effects of the Brachyury (T) mutation on morphogenetic movement in the mouse embryo. Dev. Biol. 87:242–248.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1991

Authors and Affiliations

  • Norio Nakatsuji
    • 1
  • Koichiro Hashimoto
    • 1
  1. 1.Division of Developmental BiologyMeiji Institute of Health ScienceOdawaraJapan

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