Pediatric Surgery International

, Volume 29, Issue 1, pp 13–18 | Cite as

ROCK inhibitor (Y-27632) disrupts somitogenesis in chick embryos

  • Johannes W. Duess
  • Naho Fujiwara
  • Nicolae Corcionivoschi
  • Prem Puri
  • Jennifer Thompson
Original Article



In chick embryos, administration of cadmium (Cd) induces ventral body wall defects (VBWD) similar to human omphalocele. It has been shown that failure of proper VBW formation may be due to disruption of somite development during early embryogenesis. In the VBWD chick model, Cd causes abnormal cell death in the somitic region resulting in improperly developed somites and tortuosity of the neural tube. However, the exact molecular mechanisms leading to VBWD still remain unclear. Wnt signaling is crucial during embryogenesis and plays a key role in normal somite formation. The Rho-associated coiled-coil containing protein kinase (ROCK) is involved in the non-canonical Wnt pathway which controls actin cytoskeleton assembly and cell contractility, and contributes to several developmental processes including somitogenesis. ROCK gene expression levels have recently been reported to be significantly decreased in the Cd-induced VBWD chick model. We designed this study to investigate the hypothesis that administration of ROCK inhibitor (Y-27632) in the absence of Cd disrupts somitogenesis and could contribute to the development of VBWD during early embryogenesis.


After 60 h of incubation chick embryos were transferred from eggs to culture dishes containing 20 μM of Y-27632 for experimental group (Y-27, n = 22) or chick saline for controls (n = 14). Following 24 h in the incubator they were assessed for stage development and gross abnormalities in morphology using the dissecting microscope. Western blot was performed to confirm Y-27632 inhibition of ROCK downstream signaling using an antibody against phosphorylated cofilin-2.


20 (90.9 %) embryos from Y-27 group and all controls were alive at examination. Morphological abnormalities were detected in 14 (70 %) Y-27 embryos. Somites appeared improperly developed, flattened in the cranio-caudal direction, and elongated in transverse direction in relation to controls. Chick embryos in Y-27 also presented with tortuosity of the neural tube in the lumbosacral region. Western blot analysis showed inhibition of cofilin-2 phosphorylation in affected embryos in comparison to controls.


Our study provides evidence that ROCK inhibitor results in the disruption of normal somitogenesis in chick embryos which may contribute to the failure of fusion of the anterior abdominal wall causing VBWD.


Y-27632 ROCK Wnt Chick embryo Ventral body wall defect 


  1. 1.
    Williams T (2008) Animal models of ventral body wall closure defects: a personal perspective on gastroschisis. Am J Med Genet C Semin Med Genet C 148(3):186–191CrossRefGoogle Scholar
  2. 2.
    Van Dorp DR, Malleis JM, Sullivan BP, Klein MD (2010) Teratogens inducing congenital abdominal wall defects in animal models. Pediatr Surg Int 26(2):127–139PubMedCrossRefGoogle Scholar
  3. 3.
    Sadler TW (2010) The embryologic origin of ventral body wall defects. Semin Pediatr Surg 19(3):209–214PubMedCrossRefGoogle Scholar
  4. 4.
    Ghorab H, Thompson J (2012) Understanding the etiology of ventral body wall defects. BMC Proc 6(Suppl 4):O10CrossRefGoogle Scholar
  5. 5.
    Ledbetter DJ (2006) Gastroschisis and omphalocele. Surg Clin North Am 86(2):249–260, viiGoogle Scholar
  6. 6.
    Stoll C, Alembik Y, Dott B, Roth MP (2008) Omphalocele and gastroschisis and associated malformations. Am J Med Genet A 146(10):1280–1285Google Scholar
  7. 7.
    Hamburger V (1951) Hamilton HL (1992) A series of normal stages in the development of the chick embryo. Dev Dyn 195(4):231–272CrossRefGoogle Scholar
  8. 8.
    Nagaya M, Kato J, Niimi N, Tanaka S (2000) Lordosis of lumbar vertebrae in omphalocele: an important factor in regulating abdominal cavity capacity. J Pediatr Surg 35(12):1782–1785PubMedCrossRefGoogle Scholar
  9. 9.
    Cullinane J, Bannigan J, Thompson J (2009) Cadmium teratogenesis in the chick: period of vulnerability using the early chick culture method, and prevention by divalent cations. Reprod Toxicol 28(3):335–341PubMedCrossRefGoogle Scholar
  10. 10.
    Sadler TW, Feldkamp ML (2008) The embryology of body wall closure: relevance to gastroschisis and other ventral body wall defects. Am J Med Genet C Semin Med Genet 148C(3):180–185PubMedCrossRefGoogle Scholar
  11. 11.
    Johnson ML, Rajamannan N (2006) Diseases of Wnt signaling. Rev Endocr Metab Disord 7(1–2):41–49PubMedGoogle Scholar
  12. 12.
    Stockdale FE, Nikovits W Jr, Christ B (2000) Molecular and cellular biology of avian somite development. Dev Dyn 219(3):304–321PubMedCrossRefGoogle Scholar
  13. 13.
    Montcouquiol M, Crenshaw EB 3rd, Kelley MW (2006) Noncanonical Wnt signaling and neural polarity. Annu Rev Neurosci 29:363–386PubMedCrossRefGoogle Scholar
  14. 14.
    Wang H, Lee Y, Malbon CC (2004) PDE6 is an effector for the Wnt/Ca2+/cGMP-signalling pathway in development. Biochem Soc Trans 32(Pt 5):792–796PubMedGoogle Scholar
  15. 15.
    Kang YJ, Park HJ, Chung HJ, Min HY, Park EJ, Lee MA, Shin Y, Lee SK (2012) Wnt/beta-catenin signaling mediates the antitumor activity of magnolol in colorectal cancer cells. Mol Pharmacol 82(2):168–177PubMedCrossRefGoogle Scholar
  16. 16.
    Komiya Y, Habas R (2008) Wnt signal transduction pathways. Organogenesis 4(2):68–75PubMedCrossRefGoogle Scholar
  17. 17.
    Schmidt C, McGonnell I, Allen S, Patel K (2008) The role of Wnt signalling in the development of somites and neural crest. Adv Anat Embryol Cell Biol 195:1–64PubMedCrossRefGoogle Scholar
  18. 18.
    Shi J, Wei L (2007) Rho kinase in the regulation of cell death and survival. Arch Immunol Ther Exp (Warsz) 55(2):61–75CrossRefGoogle Scholar
  19. 19.
    Kohn AD, Moon RT (2005) Wnt and calcium signaling: beta-catenin-independent pathways. Cell Calcium 38(3–4):439–446PubMedCrossRefGoogle Scholar
  20. 20.
    Riento K, Ridley AJ (2003) Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 4(6):446–456PubMedCrossRefGoogle Scholar
  21. 21.
    Shimizu Y, Thumkeo D, Keel J, Ishizaki T, Oshima H, Oshima M, Noda Y, Matsumura F, Taketo MM, Narumiya S (2005) ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles. J Cell Biol 168(6):941–953PubMedCrossRefGoogle Scholar
  22. 22.
    Thumkeo D, Shimizu Y, Sakamoto S, Yamada S, Narumiya S (2005) ROCK-I and ROCK-II cooperatively regulate closure of eyelid and ventral body wall in mouse embryo. Genes Cells 10(8):825–834PubMedCrossRefGoogle Scholar
  23. 23.
    Doi T, Puri P, Bannigan J, Thompson J (2008) Downregulation of ROCK-I and ROCK-II gene expression in the cadmium-induced ventral body wall defect chick model. Pediatr Surg Int 24(12):1297–1301PubMedCrossRefGoogle Scholar
  24. 24.
    Scott RW, Olson MF (2007) LIM kinases: function, regulation and association with human disease. J Mol Med (Berl) 85(6):555–568CrossRefGoogle Scholar
  25. 25.
    Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S (1997) Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389(6654):990–994PubMedCrossRefGoogle Scholar
  26. 26.
    Wei L, Roberts W, Wang L, Yamada M, Zhang S, Zhao Z, Rivkees SA, Schwartz RJ, Imanaka-Yoshida K (2001) Rho kinases play an obligatory role in vertebrate embryonic organogenesis. Development 128(15):2953–2962PubMedGoogle Scholar
  27. 27.
    Chapman SC, Collignon J, Schoenwolf GC, Lumsden A (2001) Improved method for chick whole-embryo culture using a filter paper carrier. Dev Dyn 220(3):284–289PubMedCrossRefGoogle Scholar
  28. 28.
    Darnell DK, Schoenwolf GC (2000) Culture of avian embryos. Methods Mol Biol 135:31–38PubMedGoogle Scholar
  29. 29.
    Thompson JM, Bannigan JG (2007) Omphalocele induction in the chick embryo by administration of cadmium. J Pediatr Surg 42(10):1703–1709PubMedCrossRefGoogle Scholar
  30. 30.
    Burke AC, Nowicki JL (2003) A new view of patterning domains in the vertebrate mesoderm. Dev Cell 4(2):159–165PubMedCrossRefGoogle Scholar
  31. 31.
    Thompson J, Bannigan J (2001) Effects of cadmium on formation of the ventral body wall in chick embryos and their prevention by zinc pretreatment. Teratology 64(2):87–97PubMedCrossRefGoogle Scholar
  32. 32.
    Thompson J, Hipwell E, Loo HV, Bannigan J (2005) Effects of cadmium on cell death and cell proliferation in chick embryos. Reprod Toxicol 20(4):539–548PubMedCrossRefGoogle Scholar
  33. 33.
    Freese JL, Pino D, Pleasure SJ (2010) Wnt signaling in development and disease. Neurobiol Dis 38(2):148–153PubMedCrossRefGoogle Scholar
  34. 34.
    Kuhl M, Sheldahl LC, Park M, Miller JR, Moon RT (2000) The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet 16(7):279–283PubMedCrossRefGoogle Scholar
  35. 35.
    Correia KM, Conlon RA (2000) Surface ectoderm is necessary for the morphogenesis of somites. Mech Dev 91(1–2):19–30PubMedCrossRefGoogle Scholar
  36. 36.
    Liao JK, Seto M, Noma K (2007) Rho kinase (ROCK) inhibitors. J Cardiovasc Pharmacol 50(1):17–24PubMedCrossRefGoogle Scholar
  37. 37.
    Thompson J, Wong L, Lau PS, Bannigan J (2008) Adherens junction breakdown in the periderm following cadmium administration in the chick embryo: distribution of cadherins and associated molecules. Reprod Toxicol 25(1):39–46PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Johannes W. Duess
    • 1
    • 2
  • Naho Fujiwara
    • 1
    • 2
  • Nicolae Corcionivoschi
    • 1
  • Prem Puri
    • 1
    • 2
  • Jennifer Thompson
    • 1
    • 2
  1. 1.National Children’s Research CentreOur Lady’s Children’s HospitalDublinIreland
  2. 2.School of Medicine and Medical Science and Conway Institute of Biomolecular and Biomedical ResearchUniversity College DublinDublinIreland

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