Downregulation of FGFRL1 Contributes to the Development of the Diaphragmatic Defect in the Nitrofen Model of Congenital Diaphragmatic Hernia

Abstract

The nitrofen model of Congenital Diaphragmatic Hernia (CDH) displays a diaphragmatic defect of the Bochdalek-type and has been widely used to investigate the pathogenesis of CDH. However, the exact pathomechanism of the diaphragmatic defect is still poorly understood. Fibroblast growth factor (FGF) receptor-like 1 (FGFRL1), a member of the FGF receptor family, plays a key role in physiological diaphragmatic development. FGFRL1 is expressed in the fetal diaphragm at low levels in early gestation and its expression steadily increases, becoming most pronounced in later gestational stages. It has been reported that FGFRL1 homozygous null mice have thin, partially amuscular diaphragms and die at birth due to respiratory failure. The aim of this study was to investigate the hypothesis that FGFRL1 gene expression in the diaphragm is downregulated during the later gestational stages in the nitrofen CDH model. Timed pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9 of gestation (D9). Cesarean section was performed on D18 or D21. Fetal diaphragms (n=40) were micro-dissected and divided into CDH group and controls. Total RNA was extracted from the diaphragms and the mRNA levels of FGFRL1 were determined using real-time PCR. Immunohistochemistry was performed to evaluate diaphragmatic protein expression of FGFRL1. Student's t-test and Mann-Whitney test were used, where appropriate. Statistical significance was considered for p<0.05. Relative mRNA expression levels of FGFRL1 were significantly decreased in the CDH group compared to controls on D18 (3.63 ± 1.65 vs. 6.04 ± 3.12, p<0.05) and D21 (1.36 ± 1.01 vs. 2.57 ± 1.34, p<0.05). Immunoreactivity of FGFRL1 was markedly decreased in the diaphragms of the CDH group compared to controls on D18 and D21. Our data provide strong evidence that downregulation of the FGFRL1 gene during the late stages of gestation may contribute to the development of the diaphragmatic defect in nitrofen-induced CDH.