Mesenchymal expression of the FRAS1/FREM2 gene unit is decreased in the developing fetal diaphragm of nitrofen-induced congenital diaphragmatic hernia

Purpose

Developmental mutations that inhibit normal formation of extracellular matrix (ECM) in fetal diaphragms have been identified in congenital diaphragmatic hernia (CDH). FRAS1 and FRAS1-related extracellular matrix 2 (FREM2), which encode important ECM proteins, are secreted by mesenchymal cells during diaphragmatic development. The FRAS1/FREM2 gene unit has been shown to form a ternary complex with FREM1, which plays a crucial role during formation of human and rodent diaphragms. Furthermore, it has been demonstrated that the diaphragmatic expression of FREM1 is decreased in the nitrofen-induced CDH model. We hypothesized that FRAS1 and FREM2 expression is decreased in the developing diaphragms of fetal rats with nitrofen-induced CDH.

Methods

Pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on D13, D15 and D18. Microdissected diaphragms were divided into nitrofen-exposed/CDH and control samples (n = 12 per time-point and experimental group, respectively). Diaphragmatic gene expression levels of FRAS1 and FREM2 were analyzed by qRT-PCR. Immunofluorescence double staining for FRAS1 and FREM2 was combined with the mesenchymal marker GATA4 in order to evaluate protein expression and localization in pleuroperitoneal folds (PPFs) and fetal diaphragmatic tissue.

Results

Relative mRNA expression of FRAS1 and FREM2 were significantly reduced in PPFs of nitrofen-exposed fetuses on D13 (1.76 ± 0.86 vs. 3.09 ± 1.15; p < 0.05 and 0.47 ± 0.26 vs. 0.82 ± 0.36; p < 0.05), developing diaphragms of nitrofen-exposed fetuses on D15 (1.45 ± 0.80 vs. 2.63 ± 0.84; p < 0.05 and 0.41 ± 0.16 vs. 1.02 ± 0.49; p < 0.05) and fully muscularized diaphragms of CDH fetuses on D18 (1.35 ± 0.75 vs. 2.32 ± 0.92; p < 0.05 and 0.37 ± 0.24 vs. 0.70 ± 0.32; p < 0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished FRAS1 and FREM2 immunofluorescence in diaphragmatic mesenchyme, which was associated with reduced proliferation of mesenchymal cells in nitrofen-exposed PPFs and fetal CDH diaphragms on D13, D15 and D18 compared to controls.

Conclusion

Decreased mesenchymal expression of FRAS1 and FREM2 in the nitrofen-induced CDH model may cause failure of the FRAS1/FREM2 gene unit to activate FREM1 signaling, disturbing the formation of diaphragmatic ECM and thus contributing to the development of diaphragmatic defects in CDH.