Eur J Pediatr Surg 2019; 29(01): 113-119
DOI: 10.1055/s-0038-1675774
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Ephrin-B1, -B2, and -B4 Expression is Decreased in Developing Diaphragms and Lungs of Fetal Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia

Toshiaki Takahashi
1  National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
,
Florian Friedmacher
1  National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
,
Julia Zimmer
1  National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
2  Department of Pediatric Surgery, Medizinische Hochschule Hannover Zentrum Chirurgie, Hannover, Germany
,
Prem Puri
1  National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
› Author Affiliations
Funding This research was supported by the National Children's Research Centre and the Children's Medical and Research Foundation.
Further Information

Publication History

11 May 2018

23 September 2018

Publication Date:
23 November 2018 (online)

Abstract

Introduction Congenital diaphragmatic hernia (CDH) is assumed to originate from a malformation of the amuscular mesenchymal component of the primordial diaphragm. Mutations in ephrin-B1, a membrane protein that is expressed by mesenchymal cells, have been found in newborn infants with CDH and associated pulmonary hypoplasia (PH), highlighting its important role during diaphragmatic and airway development. Ephrin-B1, -B2, and -B4 are expressed in fetal rat lungs and have been identified as key players during lung branching morphogenesis. We hypothesized that diaphragmatic and pulmonary expression of ephrin-B1, -B2, and -B4 is decreased in the nitrofen-induced CDH model.

Materials and Methods Time-mated rats received nitrofen or vehicle on day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 72) were harvested on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Ephrin-B1, -B2, and -B4 gene expression was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for ephrin-B1, -B2, and -B4 was combined with mesenchymal and epithelial markers (Gata-4/Fgf-10 and calcitonin gene-related peptide) to evaluate protein expression/localization.

Results Ephrin-B1, -B2, and -B4 gene expression was significantly reduced in pleuroperitoneal folds/primordial lungs (D13), developing diaphragms/lungs (D15), and fully muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal laser scanning microscopy demonstrated markedly diminished ephrin-B1 immunofluorescence in diaphragmatic and pulmonary mesenchyme of nitrofen-exposed fetuses on D13, D15, and D18 compared with controls, whereas ephrin-B2 and -B4 expression was mainly decreased in distal airway epithelium.

Conclusion Decreased ephrin-B1, -B2, and -B4 expression may disrupt diaphragmatic development and lung branching morphogenesis by interfering with epithelial–mesenchymal interactions, thus causing diaphragmatic defects and PH.