Skip to main content

Spatiotemporal Mechanical Variation Reveals Critical Role for Rho Kinase During Primitive Streak Morphogenesis

Abstract

Large-scale morphogenetic movements during early embryo development are driven by complex changes in biochemical and biophysical factors. Current models for amniote primitive streak morphogenesis and gastrulation take into account numerous genetic pathways but largely ignore the role of mechanical forces. Here, we used atomic force microscopy (AFM) to obtain for the first time precise biomechanical properties of the early avian embryo. Our data reveal that the primitive streak is significantly stiffer than neighboring regions of the epiblast, and that it is stiffer than the pre-primitive streak epiblast. To test our hypothesis that these changes in mechanical properties are due to a localized increase of actomyosin contractility, we inhibited actomyosin contractility via the Rho kinase (ROCK) pathway using the small-molecule inhibitor Y-27632. Our results using several different assays show the following: (1) primitive streak formation was blocked; (2) the time-dependent increase in primitive streak stiffness was abolished; and (3) convergence of epiblast cells to the midline was inhibited. Taken together, our data suggest that actomyosin contractility is necessary for primitive streak morphogenesis, and specifically, ROCK plays a critical role. To better understand the underlying mechanisms of this fundamental process, future models should account for the findings presented in this study.

This is a preview of subscription content, access via your institution.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

References

  1. Agero, U., J. A. Glazier, and M. Hosek. Bulk elastic properties of chicken embryos during somitogenesis. Biomed. Eng. Online 9:19, 2010.

    PubMed  Article  Google Scholar 

  2. Allison, P. D. Missing data (Sage University Papers Series on Quantitative Applications in the Social Sciences, 07–136). Thousand Oaks, CA: Sage Publications, Inc., pp. 27–36, 2002.

  3. Alonso, J. L., and W. H. Goldmann. Feeling the forces: atomic force microscopy in cell biology. Life Sci. 72:2553–2560, 2003.

    PubMed  Article  CAS  Google Scholar 

  4. An, S. S., R. E. Laudadio, J. Lai, R. A. Rogers, and J. J. Fredberg. Stiffness changes in cultured airway smooth muscle cells. Am. J. Physiol. Cell Physiol. 283:C792–C801, 2002.

    PubMed  CAS  Google Scholar 

  5. Bao, G., and S. Suresh. Cell and molecular mechanics of biological materials. Nat. Mater. 2:715–725, 2003.

    PubMed  Article  CAS  Google Scholar 

  6. Chapman, S. C., J. Collignon, G. C. Schoenwolf, and A. Lumsden. Improved method for chick whole-embryo culture using a filter paper carrier. Dev. Dyn. 220:284–289, 2001.

    PubMed  Article  CAS  Google Scholar 

  7. Chuai, M., and C. J. Weijer. The mechanisms underlying primitive streak formation in the chick embryo. Curr. Top. Dev. Biol. 81:135–156, 2008.

    PubMed  Article  Google Scholar 

  8. Chuai, M., and C. J. Weijer. Who moves whom during primitive streak formation in the chick embryo. HFSP J. 3:71–76, 2009.

    PubMed  Article  CAS  Google Scholar 

  9. Chuai, M., and C. J. Weijer. Regulation of cell migration during chick gastrulation. Curr. Opin. Genet. Dev. 19:343–349, 2009.

    PubMed  Article  CAS  Google Scholar 

  10. Chuai, M., W. Zeng, X. Yang, V. Boychenko, J. A. Glazier, and C. J. Weijer. Cell movement during chick primitive streak formation. Dev. Biol. 296:137–149, 2006.

    PubMed  Article  CAS  Google Scholar 

  11. Conte, V., J. J. Munoz, B. Baum, and M. Miodownik. Robust mechanisms of ventral furrow invagination require the combination of cellular shape changes. Phys. Biol. 6:016010, 2009.

    PubMed  Article  Google Scholar 

  12. Czirók, A., P. Rupp, B. Rongish, and C. Little. Multi-field 3-d scanning light microscopy of early embryogenesis. J. Microsc. 206:209–217, 2002.

    PubMed  Article  Google Scholar 

  13. Davidson, L., G. Oster, R. Keller, and M. A. Koehl. Measurements of mechanical properties of the blastula wall reveal which hypothesized mechanisms of primary invagination are physically plausible in the sea urchin Strongylocentrotus purpuratus. Dev. Biol. 209:221–238, 1999.

    PubMed  Article  CAS  Google Scholar 

  14. DeJonge, M., D. Burchfield, B. Bloom, M. Duenas, W. Walker, M. Polak, E. Jung, D. Millard, R. Schelonka, F. Eyal, A. Morris, B. Kapik, D. Roberson, K. Kesler, J. Patti, and S. Hetherington. Clinical trial of safety and efficacy of INH-A21 for the prevention of nosocomial staphylococcal bloodstream infection in premature infants. J. Pediatr. 151:260–265, 265 e261, 2007.

    Google Scholar 

  15. Donders, A. R., G. J. van der Heijden, T. Stijnen, and K. G. Moons. Review: a gentle introduction to imputation of missing values. J. Clin. Epidemiol. 59:1087–1091, 2006.

    PubMed  Article  Google Scholar 

  16. Eyal-Giladi, H., and S. Kochav. From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. I. General morphology. Dev. Biol. 49:321–337, 1976.

    PubMed  Article  CAS  Google Scholar 

  17. Hamburger, V., and H. Hamilton. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49–92, 1951.

    Article  Google Scholar 

  18. Hardin, J., and R. Keller. The behaviour and function of bottle cells during gastrulation of Xenopus laevis. Development 103:211–230, 1988.

    PubMed  CAS  Google Scholar 

  19. Hutter, J. L., and J. Bechhoefer. Calibration of atomic force microscope tips. Rev. Sci. Instrum. 64:1868–1873, 1993.

    Article  CAS  Google Scholar 

  20. Johnson, K. Contact Mechanics. Cambridge: Cambridge University Press, 1985.

    Google Scholar 

  21. Johnson, K. L., K. Kendall, and A. D. Roberts. Surface energy and the contact of elastic solids. Proc. R. Soc. Lond. A. Math. Phys. Sci. 324:301–313, 1971.

    Article  CAS  Google Scholar 

  22. Lee, J. Y., and R. M. Harland. Actomyosin contractility and microtubules drive apical constriction in xenopus bottle cells. Dev. Biol. 311:40–52, 2007.

    PubMed  Article  CAS  Google Scholar 

  23. Moore, S., R. Keller, and M. Koehl. The dorsal involuting marginal zone stiffens anisotropically during its convergent extension in the gastrula of Xenopus laevis. Development 121:3131–3140, 1995.

    PubMed  CAS  Google Scholar 

  24. Nakaya, Y., E. W. Sukowati, Y. Wu, and G. Sheng. RhoA and microtubule dynamics control cell-basement membrane interaction in EMT during gastrulation. Nat. Cell Biol. 10:765–775, 2008.

    PubMed  Article  CAS  Google Scholar 

  25. New, D. A. T. A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morphol. 3:320–331, 1955.

    Google Scholar 

  26. Radmacher, M. Measuring the elastic properties of biological samples with the AFM. IEEE Eng. Med. Biol. Mag. 16:47–57, 1997.

    PubMed  Article  CAS  Google Scholar 

  27. Rupp, P., B. Rongish, A. Czirók, and C. Little. Culturing of avian embryos for time-lapse imaging. Biotechniques 34:274–278, 2003.

    PubMed  CAS  Google Scholar 

  28. Sandersius, S. A., M. Chuai, C. J. Weijer, and T. J. Newman. A ‘chemotactic dipole’ mechanism for large-scale vortex motion during primitive streak formation in the chick embryo. Phys. Biol. 8:045008, 2011.

    PubMed  Article  CAS  Google Scholar 

  29. Sawyer, J. M., J. R. Harrell, G. Shemer, J. Sullivan-Brown, M. Roh-Johnson, and B. Goldstein. Apical constriction: a cell shape change that can drive morphogenesis. Dev. Biol. 341:5–19, 2010.

    PubMed  Article  CAS  Google Scholar 

  30. Trinkaus, J. Cells into Organs: The Forces that Shape the Embryo. Englewood Cliffs, NJ: Prentice-Hall, Inc., 1984.

    Google Scholar 

  31. Voiculescu, O., F. Bertocchini, L. Wolpert, R. E. Keller, and C. D. Stern. The amniote primitive streak is defined by epithelial cell intercalation before gastrulation. Nature 449:1049–1052, 2007.

    PubMed  Article  CAS  Google Scholar 

  32. Wang, N., I. Tolic-Norrelykke, J. Chen, S. Mijailovich, J. Butler, J. Fredberg, and D. Stamenovic. Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells. Am. J. Physiol. Cell Physiol. 282:C606–C616, 2002.

    PubMed  CAS  Google Scholar 

  33. Wei, L., K. Imanaka-Yoshida, L. Wang, S. Zhan, M. Schneider, F. DeMayo, and R. Schwartz. Inhibition of Rho family GTPases by Rho GDP dissociation inhibitor disrupts cardiac morphogenesis and inhibits cardiomyocyte proliferation. Development 129:1705–1714, 2002.

    PubMed  CAS  Google Scholar 

  34. Xu, W., N. Chahine, and T. Sulchek. Extreme hardening of PDMs thin films due to high compressive strain and confined thickness. Langmuir 27:8470–8477, 2011.

    PubMed  Article  CAS  Google Scholar 

  35. Zamir, E., and L. Taber. On the effects of residual stress in microindentation tests of soft tissue structures. J. Biomech. Eng. 126:276–283, 2004.

    PubMed  Article  Google Scholar 

  36. Zamir, E. A., B. J. Rongish, and C. D. Little. The ECM moves during primitive streak formation—computation of ECM versus cellular motion. PLoS Biol. 6:e247, 2008.

    PubMed  Article  Google Scholar 

  37. Zamir, E. A., V. Srinivasan, R. Perucchio, and L. A. Taber. Mechanical asymmetry in the embryonic chick heart during looping. Ann. Biomed. Eng. 31:1327–1336, 2003.

    PubMed  Article  Google Scholar 

  38. Zamir, E. A., and L. A. Taber. Material properties and residual stress in the stage 12 chick heart during cardiac looping. J. Biomech. Eng. 126:823–830, 2004.

    PubMed  Article  Google Scholar 

  39. Zhou, J., H. Y. Kim, and L. A. Davidson. Actomyosin stiffens the vertebrate embryo during crucial stages of elongation and neural tube closure. Development 136:677–688, 2009.

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by NSF Grant #1000604.

Conflict of interest

The authors have no conflict of interest to report.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Evan Zamir.

Additional information

Associate Editor Eric M. Darling oversaw the review of this article.

Wenwei Xu and Drew Owen contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (AVI 1676 kb)

Supplementary material 2 (AVI 1256 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Henkels, J., Oh, J., Xu, W. et al. Spatiotemporal Mechanical Variation Reveals Critical Role for Rho Kinase During Primitive Streak Morphogenesis. Ann Biomed Eng 41, 421–432 (2013). https://doi.org/10.1007/s10439-012-0652-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-012-0652-y

Keywords

  • Atomic force microscopy
  • Gastrulation
  • Actomyosin contractility
  • Development
  • Y-27632
  • Time-lapse imaging
  • Biomechanics