Pediatric Surgery International

, Volume 29, Issue 2, pp 165–170 | Cite as

Disturbance of SHH signalling pathway during early embryogenesis in the cadmium-induced omphalocele chick model

  • Naho Fujiwara
  • Johannes Duess
  • Prem Puri
  • Jennifer Thompson
Original Article



Administration of cadmium (Cd) after 60 h (H) incubation induces ventral body wall defect (VBWD) similar to the omphalocele phenotype in the chick embryo. In this model, the earliest histological changes have been observed in somites commencing at 4-h post-treatment (4H). The molecular mechanism by which Cd acts in this critical period of embryogenesis still remains unclear. Sonic hedgehog (SHH) signalling plays an important role in vertebrate development, including somitogenesis and thus ventral body wall formation. Patched (PTCH), a cell membrane receptor for SHH, is expressed in somites and Patched knockout mice display somite dysfunction. Another transmembrane receptor, Smoothened (SMO), is also expressed in somites and transduces the SHH signal regulated by PTCH. We designed this study to test the hypothesis that SHH signalling is downregulated during the critical period of early embryogenesis in the Cd-induced omphalocele chick model.


After 60 h of incubation, chicks were exposed to either chick saline or 50 μL of 50 μM cadmium acetate and divided into two groups: control and Cd (n = 24 for each group). Chicks were harvested 1, 4, and 8 h post-treatment. Real-time RT-PCR was performed to evaluate the relative mRNA expression level of SHH, PTCH and SMO. Immunofluorescence confocal microscopy was then performed to evaluate protein expression/distribution of SHH, PTCH and SMO.


The relative mRNA expression levels of SHH, PTCH and SMO were significantly downregulated in the Cd group compared to controls at 4H post treatment, whereas, there were no significant differences at the other time points. Immunohistochemistry revealed that the intensity of SHH, PTCH and SMO was markedly diminished at 4 h in Cd-treated embryos compared to controls.


Disturbance of the SHH signalling pathway as evidenced by SHH, PTCH and SMO downregulation during the narrow window of early embryogenesis may result in somite maldevelopment, contributing to the omphalocele phenotype in the Cd chick model.


Shh pathway Somitogenesis Cadmium Chick embryo Omphalocele 


  1. 1.
    Brewer S, Williams T (2004) Finally, a sense of closure? Animal model of human ventral body wall defects. BioEssays 26:1307–1321PubMedCrossRefGoogle Scholar
  2. 2.
    Brisic I, Clementi M, Hausler M et al (2001) Evaluation of prenatal ultrasound diagnosis of fetal abdominal wall defects by 19 European registries. Ultrasound Obstet Gynecol 18:309–316CrossRefGoogle Scholar
  3. 3.
    Thompson JM, Bannigan JG (2001) Effects of cadmium on formation of the ventral body wall in chick embryos and their prevention by zinc pre-treatment. Teratology 64:87–97PubMedCrossRefGoogle Scholar
  4. 4.
    Thompson J, Hipwell E, Loo HV et al (2005) Effects of cadmium on cell death and cell proliferation in chick embryos. Reprod Toxicol 20:539–548PubMedCrossRefGoogle Scholar
  5. 5.
    Thompson JM, Bannigan JG (2007) Omphalocele induction in the chick embryo by administration of cadmium. J Pediatr Surg 42:1703–1709PubMedCrossRefGoogle Scholar
  6. 6.
    Nagaya M, Kato J, Niimi N et al (2000) Lordosis of lumber vertebrae in omphalocele: an important factor in regulating abdominal cavity capacity. J Pediatr Surg 35:1782–1785PubMedCrossRefGoogle Scholar
  7. 7.
    Doi T, Prem P, Bannigan J et al (2012) EphB2/B3 gene expression is down-regulated during early embryogenesis in the cadmium-induced omphalocele chick model. J Pediatr Surg 47:920–924PubMedCrossRefGoogle Scholar
  8. 8.
    Doi T, Prem P, Bannigan J et al (2011) Altered PITX2 and LEF1 gene expression in the cadmium-induced omphalocele in the chick model. Pediatr Surg Int 27:495–499PubMedCrossRefGoogle Scholar
  9. 9.
    Doi T, Prem P, Bannigan J et al (2010) Disruption of noncanonical Wnt/CA2 + pathway in the cadmium-induced omphalocele in the chick model. J Pediatr Surg 45:1645–1649PubMedCrossRefGoogle Scholar
  10. 10.
    Rohatgi R, Scott MP (2007) Patching the gaps in hedgehog signalling. Nat Cell Biol 9(9):1005–1009PubMedCrossRefGoogle Scholar
  11. 11.
    Echelard Y, Epstein DJ, St-Jacques B et al (1993) Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75(7):1417–1430PubMedCrossRefGoogle Scholar
  12. 12.
    Krauss S, Concordet JP, Ingham PW (1993) A functionally conserved homolog of the drosophilia segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75(7):1431–1444PubMedCrossRefGoogle Scholar
  13. 13.
    Teillet MA, Watanabe Y, Jeffs P et al (1998) Sonic hedgehog is required for survival of both myogenic and chondrogenic somatic lineages. Development 125:2019–2030PubMedGoogle Scholar
  14. 14.
    Bumcrot DA, McMahon AP (1995) Somite differentiation: sonic signals somites. Curr Biol 5(6):612–614PubMedCrossRefGoogle Scholar
  15. 15.
    Johnson RL, Tabin C (1995) The long and short of hedgehog signaling. Cell 81:313–316PubMedCrossRefGoogle Scholar
  16. 16.
    Krüger M, Mennerich D, Fees S et al (2001) Sonic hedgehog is a survival factor for hypaxial muscles during mouse development. Development 128:743–752PubMedGoogle Scholar
  17. 17.
    Xiao C, Ogle SA, Schumacher MA et al (2010) Hedgehog signaling E-cadherin expression for the maintenance of the actin cytoskeleton and tight junctions. Am J Physiol Gastrointest Liver Physiol 299:G1252–G1265PubMedCrossRefGoogle Scholar
  18. 18.
    Gritli-Linde A, Bei M, Maas R et al (2002) Shh signaling within the dental epithelium is necessary for cell proliferation, growth and polarization. Development 129:5323–5337PubMedCrossRefGoogle Scholar
  19. 19.
    van den Brink GR (2007) Hedgehog signaling in development and homeostasis of the gastrointestinal tract. Physiol Rev 87(4):1343–1375PubMedCrossRefGoogle Scholar
  20. 20.
    Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49–92CrossRefGoogle Scholar
  21. 21.
    Dugan JD Jr, Lawton MT, Glaser B et al (1991) A new technique for explantation and in vitro cultivation of chicken embryos. Anat Rec 229:125–128PubMedCrossRefGoogle Scholar
  22. 22.
    Sadler TW, Feldkamp ML (2008) The embryology of body wall closure: relavance to gastroschisis and other ventral body wall defects. Am J Med Genet C Semin Med Genet 148:180–185Google Scholar
  23. 23.
    Borycki AG, Mendham L, Emerson CP Jr (1998) Control of somite patterning by sonic hedgehog and its downstream signal response genes. Development 125:777–790PubMedGoogle Scholar
  24. 24.
    Stockdale FE, Nikovits W Jr, Christ B (2000) Molecular and cellular biology of avian somite development. Dev Dyn 219:304–321PubMedCrossRefGoogle Scholar
  25. 25.
    Thompson JM, Wong L, Lau PS et al (2008) Adherens junction breakdown in the periderm following cadmium administration in the chick embryo: distribution of cadherins and associated molecules. Reprod Toxicol 25:39–46PubMedCrossRefGoogle Scholar
  26. 26.
    Doi T, Prem P, Bannigan J et al (2010) Disruption of calreticulin-mediated cellular adhesion signaling in the cadmium-induced omphalocele in the chick model. Pediatr Surg Int 26:91–95PubMedCrossRefGoogle Scholar
  27. 27.
    Brent AE, Tabin CJ (2002) Development regulation of somite derivatives: muscle, cartilage and tendon. Curr Opin Genet Dev 12:548–557PubMedCrossRefGoogle Scholar
  28. 28.
    Lum L, Beachy PA (2004) The hedgehog response network: sensors, switches, and routers. Science 304(5678):1755–1759PubMedCrossRefGoogle Scholar
  29. 29.
    Marigo V, Davey RA, Zuo Y et al (1996) Biochemical evidence that patched is the hedgehog receptor. Nature 384:176–179PubMedCrossRefGoogle Scholar
  30. 30.
    Hahn H, Christiansen J, Wicking C et al (1996) A mammalian patched homolog is expressed in target tissues of sonic hedgehog and maps to a region associated with developmental abnormalities. J Biol Chem 271(21):12125–12128PubMedCrossRefGoogle Scholar
  31. 31.
    Marcelle C, Stark MR, Bronner-Fraser M (1997) Coordinate actions of BMPs, Wnts, Shh and noggin mediate patterning of the dorsal somite. Development 124:3955–3963PubMedGoogle Scholar
  32. 32.
    Marigo V, Scott MP, Johnson RL et al (1996) Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development 122:1225–1233PubMedGoogle Scholar
  33. 33.
    Goodrich LV, Johnson RL, Milenkovic L et al (1996) Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by hedgehog. Genes Dev 10(3):301–312PubMedCrossRefGoogle Scholar
  34. 34.
    Goodrich LV, Milenković L, Higgins KM et al (1997) Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277(5329):1109–1113PubMedCrossRefGoogle Scholar
  35. 35.
    Snider P, Simmons O, Rogers R et al (2011) Notochordal and foregut abnormalities correlate with elevated neural crest apoptosis in patch embryos. Birth Defects Res A Clin Mol Teratol 91(6):551–564PubMedCrossRefGoogle Scholar
  36. 36.
    Resende TP, Ferreira M, Teillet MA et al (2010) Sonic hedgehog in temporal control of somite formation. Proc Natl Acad Sci USA 107(29):12907–12912PubMedCrossRefGoogle Scholar
  37. 37.
    Roessler E, Belloni E, Gaudenz K et al (1996) Mutations in the human sonic hedgehog gene cause holoprosencephaly. Nat Genet 14(3):357–360PubMedCrossRefGoogle Scholar
  38. 38.
    Zhang XM, Ramalho-Santos M, McMahon AP (2001) Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R asymmetry by the mouse node. Cell 106(2):781–792PubMedCrossRefGoogle Scholar
  39. 39.
    Chiang C, Litingtung Y, Lee Y et al (1996) Cyclopia and defective axial patterning in mice lacking sonic hedgehog gene function. Nature 383:407PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Naho Fujiwara
    • 1
  • Johannes Duess
    • 1
    • 2
  • Prem Puri
    • 1
    • 2
  • Jennifer Thompson
    • 2
  1. 1.The National Children’s Research CentreOur Lady’s Children HospitalDublin 12Ireland
  2. 2.School of Medicine and Medical Science, Conway Institute of Biomolecular and Biomedical ResearchUniversity College DublinDublinIreland

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