Investigation of molecular mechanisms of diaphragmatic defects in the nitrofen-induced rat model of congenital diaphragmatic hernia
Developmental mutations that inhibit normal formation of extracellular matrix (ECM) in fetal diaphragms have been identified in congenital diaphragmatic hernia (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. 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.
The first objective of this work was to investigate the morphological changes in the normal and abnormal diaphragm in the nitrofen rat model. The pleura-peritoneal folds (PPFs) in the control group were triangular-shaped structures protruding out from the lateral body wall, whereas nitrofen-exposed fetuses had an abnormal PPF structure, characterized by the absence of the dorsally projecting point of the triangular PPF.
The second objectives was to investigate the expression levels and distribution of FREM1, FRAS1 and FREM2 genes and their proteins in the normal and abnormal diaphragm. In nitrofen-exposed fetuses, relative mRNA expression of FREM1, FRAS1 and FREM2 were significantly reduced in developing diaphragms compared to controls. Confocal laser scanning microscopy revealed markedly diminished FREM1, FRAS1 and FREM2 immunofluorescence in diaphragmatic mesenchyme, which was associated with reduced proliferation of mesenchymal cells in nitrofen-exposed fetuses compared to controls.
Our results suggest that decreased mesenchymal expression of FREM1, FRAS1 and FREM2 in the nitrofen-induced CDH model may cause failure of the FREM1/FRAS1/FREM2 gene complex, disturbing the formation of diaphragmatic ECM and thus contributing to the development of diaphragmatic defects in CDH.
National Children’s Research Centre
First SupervisorProfessor Prem Puri
Second SupervisorProfessor Carlos Blanco
Third SupervisorProfessor Ray Stallings
CommentsA thesis submitted for the degree of Master of Science from the Royal College of Surgeons in Ireland in 2016.
Published CitationTakahashi T. Investigation of molecular mechanisms of diaphragmatic defects in the nitrofen-induced rat model of congenital diaphragmatic hernia [MSc Thesis]. Dublin: Royal College of Surgeons in Ireland; 2016.
- Master of Science (MSc): Research