Expression of dispatched RND transporter family member 1 is decreased in the diaphragmatic and pulmonary mesenchyme of nitrofen-induced congenital diaphragmatic hernia
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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.
KeywordsDisp-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.
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).
For this type of study informed consent was not required.
- 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
- 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
- 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