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.
Keywords
ephrin - diaphragm - lung - congenital diaphragmatic hernia - nitrofen
