Pediatric Surgery International

, Volume 27, Issue 2, pp 159–163 | Cite as

Expression of the Wilm’s tumor gene WT1 during diaphragmatic development in the nitrofen model for congenital diaphragmatic hernia

  • Jens Dingemann
  • Takashi Doi
  • Elke Ruttenstock
  • Prem Puri
Original Article



The nitrofen model of congenital diaphragmatic hernia (CDH) reproduces a typical diaphragmatic defect. However, the exact pathomechanism of CDH is still unknown. The Wilm’s tumor 1 gene (WT1) is crucial for diaphragmatic development. Mutations in WT1 associated with CDH have been described in humans. Additionally, WT1−/− mice display CDH. Furthermore, WT1 is involved in the retinoid signaling pathway, a candidate pathway for CDH. We hypothesized that diaphragmatic WT1 gene expression is downregulated during diaphragmatic development in the nitrofen CDH model.


Pregnant rats received vehicle or nitrofen on gestational day 9 (D9). Embryos were delivered on D13, D18 and D21. The pleuroperitoneal folds (PPFs) were dissected using laser capture microdissection (D13). Diaphragms of D18 and D21 were manually dissected. RNA was extracted and relative mRNA expression of WT1 was determined using real-time PCR. Immunofluorescence was performed to evaluate protein expression of WT1. Statistical significance was considered p < 0.05.


Diaphragmatic mRNA expression of WT1 was significantly decreased in the nitrofen group on D13, D18 and D21. Intensity of immunofluorescencence of WT1 was markedly decreased in the CDH diaphragms on D13, D18 and D21.


Downregulation of diaphragmatic WT1 gene expression may impair diaphragmatic development in the nitrofen CDH model.


Congenital diaphragmatic hernia Nitrofen Pleuroperitoneal folds Diaphragmatic defect WT1 


  1. 1.
    Gaxiola A, Varon J, Valladolid G (2009) Congenital diaphragmatic hernia: an overview of the etiology and current management. Acta Paediatr 98:621–627CrossRefPubMedGoogle Scholar
  2. 2.
    West SD, Wilson JM (2005) Follow up of infants with congenital diaphragmatic hernia. Semin Perinatol 29:129–133CrossRefPubMedGoogle Scholar
  3. 3.
    Moya FR, Lally KP (2005) Evidence-based management of infants with congenital diaphragmatic hernia. Semin Perinatol 29:112–117CrossRefPubMedGoogle Scholar
  4. 4.
    Clugston RD, Greer JJ (2007) Diaphragm development and congenital diaphragmatic hernia. Semin Pediatr Surg 16:94–100CrossRefPubMedGoogle Scholar
  5. 5.
    Mortell A, Montedonico S, Puri P (2006) Animal models in pediatric surgery. Pediatr Surg Int 22:111–128CrossRefPubMedGoogle Scholar
  6. 6.
    Clugston RD, Zhang W, Greer JJ (2008) Gene expression in the developing diaphragm: significance for congenital diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol 294:665–675CrossRefGoogle Scholar
  7. 7.
    Dingemann J, Doi T, Ruttenstock E, Puri P (2010) Downregulation of Fgfrl1 contributes to the development of the diaphragmatic defect in the nitrofen model for congenital diaphragmatic hernia. Eur J Pediatr Surg (in press)Google Scholar
  8. 8.
    Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D, Jaenisch R (1993) WT-1 is required for early kidney development. Cell 74:679–691CrossRefPubMedGoogle Scholar
  9. 9.
    SA MooreAW, McInnes L, Doyle M, Hecksher-Sorensen J, Hastie ND (1998) YAC transgenic analysis reveals Wilms’ tumour 1 gene activity in the proliferating coelomic epithelium, developing diaphragm and limb. Mech Dev 79:169–184CrossRefGoogle Scholar
  10. 10.
    Clugston RD, Klattig J, Englert C, Clagett-Dame M, Martinovic J, Benachi A, Greer JJ (2006) Teratogen-induced, dietary and genetic models of congenital diaphragmatic hernia share a common mechanism of pathogenesis. Am J Pathol 169:1541–1549CrossRefPubMedGoogle Scholar
  11. 11.
    Holder AM, Klaassens M, Tibboel D, de Klein A, Lee B, Scott DA (2007) Genetic factors in congenital diaphragmatic hernia. Am J Hum Genet 80:825–845CrossRefPubMedGoogle Scholar
  12. 12.
    Denamur E, Bocquet N, Baudouin V, Da Silva F, Veitia R, Peuchmaur M, Elion J, Gubler MC, Fellous M, Niaudet P, Loirat C (2000) WT1 splice-site mutations are rarely associated with primary steroid-resistant focal and segmental glomerulosclerosis. Kidney Int 57:1868–1872CrossRefPubMedGoogle Scholar
  13. 13.
    Devriendt K, Deloof E, Moerman P, Legius E, Vanhole C, de Zegher F, Proesmans W, Devlieger H (1995) Diaphragmatic hernia in Denys–Drash syndrome. Am J Med Genet 57:97–101CrossRefPubMedGoogle Scholar
  14. 14.
    Cho HY, Lee BS, Kang CH, Kim WH, Ha IS, Cheong HI, Choi Y (2006) Hydrothorax in a patient with Denys–Drash syndrome associated with a diaphragmatic defect. Pediatr Nephrol 21:1909–1912CrossRefPubMedGoogle Scholar
  15. 15.
    Suri M, Kelehan P, O’neill D, Vadeyar S, Grant J, Ahmed SF, Tolmie J, McCann E, Lam W, Smith S, Fitzpatrick D, Hastie ND, Reardon W (2007) WT1 mutations in Meacham syndrome suggest a coelomic mesothelial origin of the cardiac and diaphragmatic malformations. Am J Med Genet A 143A:2312–2320CrossRefPubMedGoogle Scholar
  16. 16.
    Armstrong JF, Pritchard-Jones K, Bickmore WA, Hastie ND, Bard JB (1993) The expression of the Wilms’ tumour gene, WT1, in the developing mammalian embryo. Mech Dev 40:85–97CrossRefPubMedGoogle Scholar
  17. 17.
    Clugston RD, Zhang W, Greer JJ (2010) Early development of the primordial mammalian diaphragm and cellular mechanisms of nitrofen-induced congenital diaphragmatic hernia. Birth Defects Res A Clin Mol Teratol 88:15–24PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Jens Dingemann
    • 1
  • Takashi Doi
    • 1
  • Elke Ruttenstock
    • 1
  • Prem Puri
    • 1
  1. 1.National Children’s Research Centre, Our Lady’s Children’s Hospital DublinIreland University College DublinDublin 12Ireland

Personalised recommendations