Pediatric Surgery International

, Volume 35, Issue 1, pp 35–40 | Cite as

Expression of dispatched RND transporter family member 1 is decreased in the diaphragmatic and pulmonary mesenchyme of nitrofen-induced congenital diaphragmatic hernia

  • Toshiaki Takahashi
  • Florian Friedmacher
  • Julia Zimmer
  • Prem PuriEmail author
Original Article



Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH) are thought to be caused by a malformation of the diaphragmatic and pulmonary mesenchyme. Dispatched RND transporter family member 1 (Disp-1) encodes a transmembrane protein that regulates the release of cholesterol and palmitoyl, which is critical for normal diaphragmatic and airway development. Disp-1 is strongly expressed in mesenchymal compartments of fetal diaphragms and lungs. Recently, Disp-1 mutations have been identified in patients with CDH. We hypothesized that diaphragmatic and pulmonary Disp-1 expression is decreased in the nitrofen-induced CDH model.


Time-mated rats received nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms and lungs were microdissected on selected endpoints D13, D15 and D18; and divided into control and nitrofen-exposed specimens (n = 12 per sample, time-point and experimental group). Diaphragmatic and pulmonary Disp-1 expression was evaluated by qRT-PCR. Immunofluorescence double staining for Disp-1 was combined with diaphragmatic and pulmonary mesenchymal markers Wt-1 and Sox-9 to localize protein expression in fetal diaphragms and lungs.


Relative mRNA levels of Disp-1 were significantly decreased in pleuroperitoneal folds/primordial lungs on D13 (0.18 ± 0.08 vs. 0.46 ± 0.41; p < 0.05/1.06 ± 0.27 vs. 1.34 ± 0.79; p < 0.05), developing diaphragms/lungs on D15 (0.18 ± 0.06 vs. 0.44 ± 0.23; p < 0.05/0.73 ± 0.36 vs. 1.16 ± 0.27; p < 0.05) and fully muscularized diaphragms/differentiated lungs on D18 (0.22 ± 0.18 vs. 0.32 ± 0.23; p < 0.05/0.56 ± 0.16 vs. 0.77 ± 0.14; p < 0.05) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished Disp-1 immunofluorescence predominately in the diaphragmatic and pulmonary mesenchyme of nitrofen-exposed fetuses on D13, D15 and D18, associated with a clear reduction of proliferating mesenchymal cells.


Decreased Disp-1 expression during diaphragmatic development and lung branching morphogenesis may interrupt mesenchymal cell proliferation, thus leading to diaphragmatic defects and PH in the nitrofen-induced CDH model.


Disp-1 Diaphragm Lung Congenital diaphragmatic hernia Pulmonary hypoplasia Nitrofen 



This research project was supported by grants from the National Children’s Research Centre and the Children’s Medical and Research Foundation, Ireland.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All animal procedures in this study were carried out according to the current guidelines for management and welfare of laboratory animals and the experimental protocol was fully approved by the local research ethics committee (REC668b) and the Department of Health and Children (Ref. B100/4378) under the Cruelty to Animals Act, 1876 (as amended by European Communities Regulations 2002 and 2005).

Informed consent

For this type of study informed consent was not required.


  1. 1.
    Balayla J, Abenhaim HA (2014) Incidence, predictors and outcomes of congenital diaphragmatic hernia: a population-based study of 32 million births in the United States. J Matern Fetal Neonatal Med 27:1438–1444CrossRefGoogle Scholar
  2. 2.
    McGivern MR, Best KE, Rankin J, Wellesley D, Greenlees R, Addor MC, Arriola L, de Walle H, Barisic I, Beres J, Bianchi F, Calzolari E, Doray B, Draper ES, Garne E, Gatt M, Haeusler M, Khoshnood B, Klungsoyr K, Latos-Bielenska A, O’Mahony M, Braz P, McDonnell B, Mullaney C, Nelen V, Queisser-Luft A, Randrianaivo H, Rissmann A, Rounding C, Sipek A, Thompson R, Tucker D, Wertelecki W, Martos C (2015) Epidemiology of congenital diaphragmatic hernia in Europe: a register-based study. Arch Dis Child Fetal Neonatal Ed 100:F137–F144CrossRefGoogle Scholar
  3. 3.
    Morini F, Capolupo I, van Weteringen W, Reiss I (2017) Ventilation modalities in infants with congenital diaphragmatic hernia. Semin Pediatr Surg 26:159–165CrossRefGoogle Scholar
  4. 4.
    Harting MT, Lally KP (2014) The congenital diaphragmatic hernia study group registry update. Semin Fetal Neonatal Med 19:370–375CrossRefGoogle Scholar
  5. 5.
    Greer JJ (2013) Current concepts on the pathogenesis and etiology of congenital diaphragmatic hernia. Respir Physiol Neurobiol 189:232–240CrossRefGoogle Scholar
  6. 6.
    Merrell AJ, Kardon G (2013) Development of the diaphragm—a skeletal muscle essential for mammalian respiration. FEBS J 280:4026–4035CrossRefGoogle Scholar
  7. 7.
    Maki JM, Sormunen R, Lippo S, Kaarteenaho-Wiik R, Soininen R, Myllyharju J (2005) Lysyl oxidase is essential for normal development and function of the respiratory system and for the integrity of elastic and collagen fibers in various tissues. Am J Pathol 167:927–936CrossRefGoogle Scholar
  8. 8.
    Hornstra IK, Birge S, Starcher B, Bailey AJ, Mecham RP, Shapiro SD (2003) Lysyl oxidase is required for vascular and diaphragmatic development in mice. J Biol Chem 278:14387–14393CrossRefGoogle Scholar
  9. 9.
    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 88:15–24Google Scholar
  10. 10.
    van Loenhout RB, Tibboel D, Post M, Keijzer R (2009) Congenital diaphragmatic hernia: comparison of animal models and relevance to the human situation. Neonatology 96:137–149CrossRefGoogle Scholar
  11. 11.
    Montedonico S, Nakazawa N, Puri P (2008) Congenital diaphragmatic hernia and retinoids: searching for an etiology. Pediatr Surg Int 24:755–761CrossRefGoogle Scholar
  12. 12.
    Noble BR, Babiuk RP, Clugston RD, Underhill TM, Sun H, Kawaguchi R, Walfish PG, Blomhoff R, Gundersen TE, Greer JJ (2007) Mechanisms of action of the congenital diaphragmatic hernia-inducing teratogen nitrofen. Am J Physiol Lung Cell Mol Physiol 293:L1079–L1087CrossRefGoogle Scholar
  13. 13.
    Kawakami T, Kawcak T, Li YJ, Zhang W, Hu Y, Chuang PT (2002) Mouse dispatched mutants fail to distribute hedgehog proteins and are defective in hedgehog signaling. Development 129:5753–5765CrossRefGoogle Scholar
  14. 14.
    Etheridge LA, Crawford TQ, Zhang S, Roelink H (2010) Evidence for a role of vertebrate Disp1 in long-range Shh signaling. Development 137:133–140CrossRefGoogle Scholar
  15. 15.
    Kantarci S, Ackerman KG, Russell MK, Longoni M, Sougnez C, Noonan KM, Hatchwell E, Zhang X, Pieretti-Vanmarcke R, Anyane-Yeboa K, Dickman P, Wilson J, Donahoe PK, Pober BR (2010) Characterization of the chromosome 1q41q42.12 region, and the candidate gene DISP1, in patients with CDH. Am J Med Genet Part A 152:2493–2504CrossRefGoogle Scholar
  16. 16.
    Herriges M, Morrisey EE (2014) Lung development: orchestrating the generation and regeneration of a complex organ. Development 141:502–513CrossRefGoogle Scholar
  17. 17.
    Short K, Hodson M, Smyth I (2013) Spatial mapping and quantification of developmental branching morphogenesis. Development 140:471–478CrossRefGoogle Scholar
  18. 18.
    Friedmacher F, Gosemann JH, Fujiwara N, Takahashi H, Hofmann A, Puri P (2013) Expression of Sproutys and SPREDs is decreased during lung branching morphogenesis in nitrofen-induced pulmonary hypoplasia. Pediatr Surg Int 29:1193–1198CrossRefGoogle Scholar
  19. 19.
    Babiuk RP, Zhang W, Clugston R, Allan DW, Greer JJ (2003) Embryological origins and development of the rat diaphragm. J Comp Neurol 455:477–487CrossRefGoogle Scholar
  20. 20.
    Merrell AJ, Ellis BJ, Fox ZD, Lawson JA, Weiss JA, Kardon G (2015) Muscle connective tissue controls development of the diaphragm and is a source of congenital diaphragmatic hernias. Nat Genet 47:496–504CrossRefGoogle Scholar
  21. 21.
    Takahashi T, Friedmacher F, Takahashi H, Hofmann AD, Puri P (2015) Kif7 expression is decreased in the diaphragmatic and pulmonary mesenchyme of nitrofen-induced congenital diaphragmatic hernia. J Pediatr Surg 50:904–907CrossRefGoogle Scholar
  22. 22.
    Takahashi T, Friedmacher F, Takahashi H, Daniel Hofmann A, Puri P (2014) Lysyl oxidase expression is decreased in the developing diaphragm and lungs of nitrofen-induced congenital diaphragmatic hernia. Eur J Pediatr Surg 25:15–19CrossRefGoogle Scholar
  23. 23.
    Heussler HS, Suri M, Tibboel D, de Klein A, Lee B, Scott DA (2007) Genetic factors in congenital diaphragmatic hernia. Am J Hum Genet 80:825–845CrossRefGoogle Scholar
  24. 24.
    Rottier R, Tibboel D (2005) Fetal lung and diaphragm development in congenital diaphragmatic hernia. Semin Perinatol 29:86–93CrossRefGoogle Scholar
  25. 25.
    Unger S, Copland I, Tibboel D (2003) Down-regulation of sonic hedgehog expression in pulmonary hypoplasia is associated with congenital diaphragmatic hernia. Am J Pathol 162(2):547–555CrossRefGoogle Scholar
  26. 26.
    Coles GL, Ackerman KG (2013) Kif7 is required for the patterning and differentiation of the diaphragm in a model of syndromic congenital diaphragmatic hernia. Proc Natl Acad Sci USA 110:E1898–E1905CrossRefGoogle Scholar
  27. 27.
    Cheung HO, Zhang X, Ribeiro A (2009) The kinesin protein Kif7 is a critical regulator of Gli transcription factors in mammalian hedgehog signaling. Sci Signal 2:ra29CrossRefGoogle Scholar
  28. 28.
    Carmona R, Canete A, Cano E, Ariza L, Rojas A, Munoz-Chapuli R (2016) Conditional deletion of WT1 in the septum transversum mesenchyme causes congenital diaphragmatic hernia in mice. Elife 19:e16009CrossRefGoogle Scholar
  29. 29.
    Paris ND, Coles GL, Ackerman KG (2015) Wt1 and beta-catenin cooperatively regulate diaphragm development in the mouse. Dev Biol 407:40–56CrossRefGoogle Scholar
  30. 30.
    Chang DR, Martinez Alanis D, Miller RK, Ji H, Akiyama H, McCrea PD, Chen J (2013) Lung epithelial branching program antagonizes alveolar differentiation. Proc Natl Acad Sci USA 110:18042–18051CrossRefGoogle Scholar
  31. 31.
    Turcatel G, Rubin N, Menke DB, Martin G, Shi W, Warburton D (2013) Lung mesenchymal expression of Sox9 plays a critical role in tracheal development. BMC Biol 11:117CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Toshiaki Takahashi
    • 1
  • Florian Friedmacher
    • 1
    • 2
  • Julia Zimmer
    • 1
  • Prem Puri
    • 1
    • 3
    Email author
  1. 1.National Children’s Research CentreOur Lady’s Children’s HospitalDublin 12Ireland
  2. 2.Department of Pediatric SurgeryThe Royal London HospitalLondonUK
  3. 3.Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical ScienceUniversity College DublinDublinIreland

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