title: Decreased expression of hepatocyte growth factor in the nitrofen model of congenital diaphragmatic hernia.
abstract: OBJECTIVE
  Pleuroperitoneal folds (PPFs) are essential for normal diaphragmatic development, representing the only source of the diaphragm's muscle connective tissue. Hepatocyte growth factor (Hgf), which is secreted in PPFs, plays a crucial role in the formation of the muscular diaphragmatic components by regulating the migration of myogenic progenitor cells into the primordial diaphragm. Hgf is also a known downstream target of Gata4 and it has been demonstrated that the expression of Hgf was significantly downregulated in PPF cells of Gata4 knockouts with congenital diaphragmatic hernia (CDH). Furthermore, mutations in PPF-derived cells have been shown to result in CDH. We hypothesized that Hgf expression is decreased in developing diaphragms of fetal rats with nitrofen-induced CDH.
  
  
  METHODS
  Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on selected time-points D13, D15 and D18. Dissected diaphragms (n = 72) were divided into control and nitrofen-exposed specimens (n = 12 per time-point and experimental group, respectively). Diaphragmatic gene expression of Hgf was analyzed by qRT-PCR. Immunofluorescence double staining for Hgf and the mesenchymal marker Gata4 or muscular progenitor marker Myogenin was performed to evaluate protein expression and localization in fetal diaphragms.
  
  
  RESULTS
  Relative mRNA expression of Hgf was significantly downregulated in PPFs of nitrofen-exposed fetuses on D13 (3.08 ± 1.46 vs. 5.24 ± 1.93; p < 0.05), developing diaphragms of nitrofen-exposed fetuses on D15 (2.01 ± 0.79 vs. 4.10 ± 1.50; p < 0.05) and fully muscularized diaphragms of nitrofen-exposed fetuses on D18 (1.60 ± 0.78 vs. 3.21 ± 1.89; p < 0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished diaphragmatic immunofluorescence of Hgf in nitrofen-exposed fetuses on D13, D15 and D18 compared to controls, which was associated with disruptions in muscle connective tissue formation and reduced myogenic progenitor cell invasion.
  
  
  CONCLUSIONS
  Decreased diaphragmatic expression of Hgf may disturb the formation of muscle connective tissue in PPFs and thus prevent essential migration of muscle progenitor cells into the developing diaphragm, leading to diaphragmatic defects in the nitrofen CDH model.
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Florian
authors.initials: F
authors.lastname: Friedmacher
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Julia
authors.initials: J
authors.lastname: Zimmer
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland. prem.puri@ncrc.ie.|Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland. prem.puri@ncrc.ie.
authors.forename: Prem
authors.initials: P
authors.lastname: Puri
mesh_terms: D000818:Animals; D003964:Diaphragm / Q000196:embryology; D004195:Disease Models, Animal; D047108:Embryonic Development; D005260:Female; D005333:Fetus / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D017228:Hepatocyte Growth Factor / Q000097:blood* / Q000235:genetics*; D065630:Hernias, Diaphragmatic, Congenital / Q000235:genetics* / Q000378:metabolism*; D010647:Phenyl Ethers; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D010647:Phenyl Ethers; D017228:Hepatocyte Growth Factor; C007350:nitrofen
keywords: Congenital diaphragmatic hernia; Diaphragm; Hepatocyte growth factor; Nitrofen