title: Fibrillin-1 Expression Is Decreased in the Diaphragmatic Muscle Connective Tissue of Nitrofen-Induced Congenital Diaphragmatic Hernia.
abstract: IntroductionDiaphragmatic morphogenesis depends on proper formation of muscle connective tissue (MCT) and underlying extracellular matrix (ECM). Fibrillin-1 is an essential ECM protein and crucial for the structural integrity of MCT in the developing diaphragm. Recently, mutations in the fibrillin-1 gene (FBN1) have been identified in cases of congenital diaphragmatic hernia (CDH), thus suggesting that alterations in FBN1 gene expression may lead to diaphragmatic defects. We designed this study to investigate the hypothesis that the diaphragmatic expression of fibrillin-1 is decreased in the MCT of nitrofen-induced CDH.Materials and MethodsTime-mated rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n= 72) were harvested on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Laser-capture microdissection was used to obtain diaphragmatic tissue cells. Gene expression levels of FBN1 were analyzed by qRT-PCR. Immunofluorescence-double-staining for fibrillin-1 and the mesenchymal marker Gata4 was performed to evaluate protein expression and localization.ResultsRelative mRNA expression of FBN1 was significantly decreased in pleuroperitoneal folds on D13 (3.39 ± 1.29 vs. 5.47 ± 1.92;p< 0.05), developing diaphragms on D15 (2.48 ± 0.89 vs. 4.03 ± 1.62;p< 0.05), and fully muscularized diaphragms on D18 (2.49 ± 0.69 vs. 3.93 ± 1.55;p< 0.05) of nitrofen-exposed fetuses compared with controls. Confocal-laser-scanning microscopy revealed markedly diminished fibrillin-1 immunofluorescence mainly in MCT, associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared with controls.ConclusionsDecreased expression of fibrillin-1 during morphogenesis of the fetal diaphragm may disrupt mesenchymal cell proliferation, causing malformed MCT and thus resulting in diaphragmatic defects in the nitrofen-induced CDH model.
authors: National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland.
authors: Toshiaki
authors: T
authors: Takahashi
authors: National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland.
authors: Florian
authors: F
authors: Friedmacher
authors: National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland.
authors: Julia
authors: J
authors: Zimmer
authors: National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland.
authors: Prem
authors: P
authors: Puri
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D003238:Connective Tissue / Q000196:embryology* / Q000378:metabolism; D003964:Diaphragm / Q000196:embryology* / Q000378:metabolism; D015536:Down-Regulation; D000071838:Fibrillin-1 / Q000235:genetics* / Q000378:metabolism; D050991:GATA4 Transcription Factor / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D065630:Hernias, Diaphragmatic, Congenital / Q000139:chemically induced / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D010647:Phenyl Ethers; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D054392:Adipokines
chemical_list: D000071838:Fibrillin-1; D050991:GATA4 Transcription Factor; D005819:Genetic Markers; D010647:Phenyl Ethers; C000605725:Fbn1 protein, rat; D054392:Adipokines; C084365:Gata4 protein, rat; C007350:nitrofen