Advertisement

Cell Fusion pp 315-334 | Cite as

Mesenchymal Cell Fusion in the Sea Urchin Embryo

  • Paul G. Hodor
  • Charles A. Ettensohn
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 475)

Summary

Mesenchymal cells of the sea urchin embryo provide a valuable experimental model for the analysis of cell–cell fusion in vivo. The unsurpassed optical transparency of the sea urchin embryo facilitates analysis of cell fusion in vivo using fluorescent markers and time-lapse three-dimensional imaging. Two populations of mesodermal cells engage in homotypic cell–cell fusion during gastrulation: primary mesenchyme cells and blastocoelar cells. In this chapter, we describe methods for studying the dynamics of cell fusion in living embryos. These methods have been used to analyze the fusion of primary mesenchyme cells and are also applicable to blastocoelar cell fusion. Although the molecular basis of cell fusion in the sea urchin has not been investigated, tools have recently become available that highlight the potential of this experimental model for integrating dynamic morphogenetic behaviors with underlying molecular mechanisms.

Key Words:

Sea urchin; embryo; gastrulation; primary mesenchyme cells; secondary mesen-chyme cells; blastocoelar cells; cell fusion; time-lapse microscopy; fluorescent dyes. 

References

  1. 1.
    Ben-Tabou de-Leon, S. and Davidson, E. H. (2007) Gene regulation: gene control network in development. Ann. Rev. Biophys. Biomol. Struct. 36, 191–212.CrossRefGoogle Scholar
  2. 2.
    Levine, M. and Davidson, E. H. (2005) Gene regulatory networks for development. Proc. Natl. Acad. Sci. U.S.A. 102, 4936–4942.CrossRefPubMedGoogle Scholar
  3. 3.
    Cameron, R. A., Rast, J. P., and Brown, C. T. (2004) Genomic resources for the study of sea urchin development. Methods Cell Biol. 74, 733–757.CrossRefPubMedGoogle Scholar
  4. 4.
    Sea Urchin Genome Sequencing Consortium (2006) The genome of the sea urchin Strongylocentrotus purpuratus. Science 314, 941–952.CrossRefGoogle Scholar
  5. 5.
    Wilt, F. H. and Ettensohn, C. A. (2007) The morphogenesis and biomineralization of the sea urchin larval skeleton, in Handbook of Biomineralization (E. Bauerlein, ed.). Wiley-VCH Press, New York, pp. 183–210.Google Scholar
  6. 6.
    Cheers, M. S. and Ettensohn, C. A. (2005) P16 is an essential regulator of skel-etogenesis in the sea urchin embryo. Dev. Biol. 283, 384–396.CrossRefPubMedGoogle Scholar
  7. 7.
    Ettensohn, C. A., Illies, M. R., Oliveri, P., and De Jong, D. L. (2003) Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-class homeodomain proteins, is an essential component of the gene network controlling skeletogenic fate specification in the sea urchin embryo. Development 130, 2917–2928.CrossRefPubMedGoogle Scholar
  8. 8.
    Illies. M. R., Peeler, M. T., Dechtiaruk, A. M., and Ettensohn, C. A. (2002) Identification and developmental expression of new biomineralization proteins in the sea urchin, Strongylocentrotus purpuratus. Dev. Genes Evol. 212, 419–431.CrossRefPubMedGoogle Scholar
  9. 9.
    Livingston, B. T., Killian, C. E., Wilt, F., Cameron, A., Landrum, M. J., Ermolaeva, O., Sapojnikov, V., Maglott, D. R., Buchanan, A. M., and Ettensohn, C. A. (2006) A genome-wide analysis of biomineralization-related proteins in the sea urchin, Strongylocentrotus purpuratus. Dev. Biol. 300, 335–348.CrossRefPubMedGoogle Scholar
  10. 10.
    Zhu, X., Mahairas, G., Illies, M., Cameron, R. A., Davidson, E. H., and Ettensohn, C. A. (2001) A large-scale analysis of mRNAs expressed by primary mesenchyme cells of the sea urchin embryo. Development 128, 2615–2627.PubMedGoogle Scholar
  11. 11.
    Oliveri, P. and Davidson, E. H. (2004) Gene regulatory network controlling embryonic specification in the sea urchin. Curr. Opin. Genet. Dev. 14, 351–360.CrossRefPubMedGoogle Scholar
  12. 12.
    Hodor, P. G. and Ettensohn, C. A. (1998) The dynamics and regulation of mesen-chymal cell fusion in the sea urchin embryo. Dev. Biol. 199, 111–124.CrossRefPubMedGoogle Scholar
  13. 13.
    Burke, R. D. and Alvarez, C. M. (1988) Development of the esophageal muscles in embryos of the sea urchin Strongylocentrotus purpuratus. Cell Tissue Res. 252, 411–417.CrossRefPubMedGoogle Scholar
  14. 14.
    Tamboline, C. R. and Burke, R. D. (1992) Secondary mesenchyme of the sea urchin embryo: ontogeny of blastocoelar cells. J. Exp. Zool. 262, 51–60.CrossRefPubMedGoogle Scholar
  15. 15.
    Kominami, T. and Takata, H. (2003) Specification of secondary mesenchymederived cells in relation to the dorso-ventral axis in sea urchin blastulae. Dev. Growth Differ. 45, 129–142.CrossRefPubMedGoogle Scholar
  16. 16.
    Ruffins, S. W. and Ettensohn, C. A. (1993) A clonal analysis of secondary mes-enchyme cell fates in the sea urchin embryo. Dev. Biol. 160, 285–288.CrossRefPubMedGoogle Scholar
  17. 17.
    Ruffins, S. W. and Ettensohn, C. A. (1996) A fate map of the vegetal plate of the sea urchin (Lytechinus variegatus) mesenchyme blastula. Development 122, 253–263.PubMedGoogle Scholar
  18. 18.
    Sweet, H. C., Gehring, M., and Ettensohn, C. A. (2002) LvDelta is a mesoderm-inducing signal in the sea urchin embryo and can endow blastomeres with organizer-like properties. Development 129, 1945–1955.PubMedGoogle Scholar
  19. 19.
    Howard-Ashby, M., Materna, S. C., Brown, C. T., Chen, L., Cameron, R. A., and Davidson, E. H. (2006) Gene families encoding transcription factors expressed inearly development of Strongylocentrotus purpuratus. Dev. Biol. 300, 90–107.CrossRefPubMedGoogle Scholar
  20. 20.
    Miller, R. N., Dalamagas, D. G., Kingsley, P. D., and Ettensohn, C. A. (1996) Expression of S9 and actin CyIIa mRNAs reveals dorso-ventral polarity and mesodermal sublineages in the vegetal plate of the sea urchin embryo. Mech. Dev. 60, 3–12.CrossRefPubMedGoogle Scholar
  21. 21.
    Rottinger, E., Besnardeau, L., and Lepage, T. (2004) A Raf/MEK/ERK signaling pathway is required for development of the sea urchin embryo micromere lineage through phosphorylation of the transcription factor Ets. Development 131, 1075–1087.CrossRefPubMedGoogle Scholar
  22. 22.
    Shoguchi, E., Tokuoka, M., and Kominami, T. (2002) In situ screening for genes expressed preferentially in secondary mesenchyme cells of sea urchin embryos. Dev. Genes Evol. 212, 407–418.CrossRefPubMedGoogle Scholar
  23. 23.
    Sweet, H. C., Hodor, P. G., and Ettensohn, C. A. (1999) The role of micromere signaling in Notch activation and mesoderm specification during sea urchin embryogenesis. Development 126, 5255–5265.PubMedGoogle Scholar
  24. 24.
    Okazaki, K. (1960) Skeleton formation of sea urchin larvae. II. Organic matrix of the spicule. Embryologia 5, 283–320.CrossRefGoogle Scholar
  25. 25.
    Okazaki, K. (1965) Skeleton formation of sea urchin larvae. V. Continuous observation of the process of matrix formation. Exp. Cell Res. 40, 585–596.CrossRefPubMedGoogle Scholar
  26. 26.
    Gustafson, T. and Wolpert, L. (1967) Cellular movement and contact in sea urchin morphogenesis. Biol. Rev. 42, 441–498.CrossRefGoogle Scholar
  27. 27.
    Gibbins, J. R., Tilney, L. G., and Porter, K. R. (1969) Microtubules in the formation and development of the primary mesenchyme in Arbacia punctulata. I. The distribution of microtubules. J. Cell Biol. 41, 201–226.CrossRefPubMedGoogle Scholar
  28. 28.
    Okazaki, K. (1975) Spicule formation by isolated micromeres of the sea urchin embryo. Am. Zool. 15, 567–581.Google Scholar
  29. 29.
    Cheers, M. S. and Ettensohn, C. A. (2004) Rapid microinjection of fertilized eggs. Methods Cell Biol. 74, 287–310.CrossRefPubMedGoogle Scholar
  30. 30.
    Foltz, K. R., Adams, N. L., and Runft, L. L. (2004) Echinoderm eggs and embryos: procurement and culture. Methods Cell Biol. 74, 39–74.CrossRefPubMedGoogle Scholar
  31. 31.
    Jaffe, L. A. and Terasaki, M. (2004) Quantitative microinjection of oocytes, eggs, and embryos. Methods Cell Biol. 74, 219–242.CrossRefPubMedGoogle Scholar
  32. 32.
    Sweet, H., Amemiya, S., Ransick, A., Minokawa, T., McClay, D. R., Wikramanayake, A., Kuraishi, R., Kiyomoto, M., Nishida, H., and Henry, J. (2004) Blastomere isolation and transplantation. Methods Cell Biol. 74, 243–271.CrossRefPubMedGoogle Scholar
  33. 33.
    Arnone, M. I., Dmochowski, I. J., and Gache, C. (2004) Using reporter genes to study cis-regulatory elements. Methods Cell Biol. 74, 621–652.CrossRefPubMedGoogle Scholar
  34. 34.
    Arnone, M. I., Bogarad, L. D., Collazo, A., Kirchhamer, C. V., Cameron, R. A., Rast, J. P., Gregorians, A., and Davidson, E. H. (1997) Green fluorescent protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae. Development 124, 4649–4659.PubMedGoogle Scholar
  35. 35.
    Arnone, M. I., Martin, E. L., and Davidson, E. H. (1998) Cis-regulation downstream of cell type specification: a single compact element controls the complex expression of the CyIIa gene in sea urchin embryos. Development 125, 1381–1395.PubMedGoogle Scholar
  36. 36.
    Harkey, M. A., Klueg, K., Sheppard, P., and Raff, R. A. (1995) Structure, expression, and extracellular targeting of PM27, a skeletal protein associated specifically with growth of the sea urchin larval spicule. Dev. Biol. 168, 549–566.CrossRefPubMedGoogle Scholar
  37. 37.
    Makabe, K. W., Kirchhamer, C. V. , Britten, R. J., and Davidson, E. H. (1995) Cis-regulatory control of the SM50 gene, an early marker of skeletogenic lineage specification in the sea urchin embryo. Development 121, 1957–1970.PubMedGoogle Scholar
  38. 38.
    Klueg, K. M., Harkey, M. A., and Raff, R. A. (1997) Mechanisms of evolutionary changes in timing, spatial expression, and mRNA processing in the msp130 gene in a direct-developing sea urchin, Heliocidaris erythrogramma. Dev. Biol. 182, 121–133.CrossRefPubMedGoogle Scholar
  39. 39.
    Martin, E. L., Consales, C., Davidson, E. H., and Arnone, M. I. (2001) Evidence for a mesodermal embryonic regulator of the sea urchin CyIIa gene. Dev. Biol. 236, 46–63.CrossRefPubMedGoogle Scholar
  40. 40.
    Yamasu, K. and Wilt, F. H. (1999) Functional organization of DNA elements regulating SM30alpha, a spicule matrix gene of sea urchin embryos. Dev. Growth Differ. 41, 81–91.CrossRefPubMedGoogle Scholar
  41. 41.
    Kiehart, D. (1982) Microinjection of echinoderm eggs: apparatus andprocedures. Methods Cell Biol. 25, 13–31.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Paul G. Hodor
    • 1
  • Charles A. Ettensohn
    • 1
  1. 1.Department of Biological SciencesCarnegie Mellon UniversityPittsburgh

Personalised recommendations