Gene Expression of FRAS1-Related Extracellular Matrix 1 Is Decreased in Nitrofen-Induced Congenital Diaphragmatic Hernia

Abstract

Introduction The origin of congenital diaphragmatic hernia (CDH) is considered to lie in a malformation of the nonmuscular primordial diaphragm. It is known that fetal diaphragmatic development requires the structural integrity of its underlying mesenchymal tissue. Developmental mutations that inhibit the formation of normal diaphragmatic mesenchyme have been shown to cause CDH. FRAS1-related extracellular matrix 1 (FREM1) plays a critical role in the development of the fetal diaphragm. It has been demonstrated that a deficiency of FREM1 can lead to CDH both in humans and mice. Furthermore, FREM1-deficient fetuses exhibit a decreased level of mesenchymal cell proliferation in their developing diaphragms. We hypothesized that FREM1 expression is decreased in developing diaphragms of fetal rats with nitrofen-induced CDH.

Material and Methods Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on selected time-points D13, D15, and D18. Dissected diaphragms (n = 72) were divided into control and nitrofen-exposed samples (n = 12 per time-point and experimental group). Diaphragmatic gene expression levels of FREM1 were analyzed by quantitative polymerase chain reaction. Immunofluorescence staining for FREM1 was combined with the mesenchymal marker GATA4 to localize FREM1 protein expression and tissue distribution in fetal diaphragms.

Results In nitrofen-exposed fetuses, relative mRNA expression of FREM1 was significantly reduced in pleuroperitoneal folds on D13 (0.30 ± 0.23 vs. 0.83 ± 0.19; p < 0.05), developing diaphragms on D15 (0.54 ± 0.22 vs. 1.19 ± 0.28; p < 0.05) and fully muscularized diaphragms on D18 (0.49 ± 0.37 vs. 0.97 ± 0.53; p < 0.05) in comparison with controls. Confocal laser scanning microscopy revealed markedly diminished diaphragmatic FREM1 immunofluorescence, which was associated with reduced proliferation of diaphragmatic mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared to controls.

Conclusions Decreased expression of FREM1 in the nitrofen-induced CDH model may disturb the formation of the diaphragmatic mesenchyme, thus contributing to the development of diaphragmatic defects.

Keywords

FRAS1-related extracellular matrix 1 - FREM1 - diaphragm - nitrofen - congenital diaphragmatic hernia

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