Decreased Expression of GATA4 in the Diaphragm of Nitrofen‐Induced Congenital Diaphragmatic Hernia

Abstract

The molecular mechanisms underlying the diaphragmatic defect in congenital diaphragmatic hernia (CDH) are still poorly understood. The transcription factor GATA4 is essential for normal development of the diaphragm. Recently, mutations in the GATA4 gene have been linked to human and rodent CDH. We hypothesized that diaphragmatic GATA4 expression is downregulated in the nitrofen CDH model. Pregnant rats received Nitrofen or vehicle on day 9 of gestation (D9). Fetuses were sacrificed on D13, D18, or D21. Pleuroperitoneal folds (n=20) and fetal diaphragms (n=40) were (micro) dissected and divided into CDH group and controls. RNA and protein were extracted. GATA4 mRNA levels were determined by real-time PCR. Protein levels were determined by ELISA and Immunohistochemistry. mRNA levels and Protein levels were significantly decreased in the CDH group compared to controls on D13 (mRNA 15.96±6.99 vs. 38.10±5.01, p<0.05), D18 (mRNA 10.45±1.84 vs. 17.68±2.11, Protein 2.59±0.06 vs. 4.58±0.35 p<0.05) and D21 (mRNA 4.31±0.83 vs. 6.87±0.88, Protein 0.16±0.08 vs. 1.26±0.49, p<0.05). Immunoreactivity of GATA4 was markedly decreased in CDH-diaphragms on D13, D18, and D21. We provide evidence for the first time that diaphragmatic expression of GATA4 is downregulated in the nitrofen model, suggesting that decreased expression of GATA4 may impair diaphragmatic development in nitrofen-induced CDH.