title: Expression of Eya1 and Six1 is decreased in distal airways of rats with experimental pulmonary hypoplasia.
abstract: OBJECTIVE
  Pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH) represents one of the major challenges in neonatal intensive care. Eyes absent 1 (Eya1) and sine oculis homebox 1 (Six1) have been identified as essential components of the gene network that regulates foetal lung development. Eya1 and Six1 are expressed in distal epithelial tips of branching airways as well as in surrounding mesenchymal cells, highlighting their important role during branching morphogenesis. Lungs of Eya1(-/-) and Six1(-/-) knockouts display PH with reduced epithelial branching, appearing to be arrested in the pseudoglandular stage. We hypothesized that Eya1 and Six1 expression is decreased in branching airways of nitrofen-induced PH.
  
  
  METHODS
  Time-mated rats received either nitrofen or vehicle on E9.5. Foetal lungs were dissected on E15.5 and divided into control and nitrofen groups, whereas lungs harvested on E18.5 were divided into controls, PH without CDH [PH(-)], and PH with CDH [PH(+)]. Pulmonary gene expression levels of Eya1 and Six1 were analyzed by quantitative real-time PCR. Immunofluorescence staining was performed to investigate Eya1 and Six1 protein expression and localization by confocal laser scanning microscopy (CLSM).
  
  
  RESULTS
  Relative mRNA expression of Eya1 and Six1 was significantly decreased in PH(-) and PH(+) on E18.5 compared to controls. CLSM confirmed markedly diminished immunofluorescence of Eya1 and Six1 in distal airway epithelium as well as in surrounding mesenchymal cells of nitrofen-induced PH on E18.5 compared to controls.
  
  
  CONCLUSIONS
  Downregulation of Eya1 and Six1 gene expression in nitrofen-induced PH suggests that decreased Eya1 and Six1 expression during the late pseudoglandular stage may interfere with epithelial branching and distal airway maturation, thus resulting in PH.
authors: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.
authors: Florian
authors: F
authors: Friedmacher
authors: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.
authors: Naho
authors: N
authors: Fujiwara
authors: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.
authors: Alejandro Daniel
authors: AD
authors: Hofmann
authors: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.
authors: Hiromizu
authors: H
authors: Takahashi
authors: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.
authors: Jan-Hendrik
authors: JH
authors: Gosemann
authors: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland; Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland. Electronic address: prem.puri@ncrc.ie.
authors: Prem
authors: P
authors: Puri
mesh_terms: D000818:Animals; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006548:Hernia, Diaphragmatic / Q000235:genetics; D065630:Hernias, Diaphragmatic, Congenital; D018398:Homeodomain Proteins / Q000235:genetics*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D008168:Lung / Q000002:abnormalities*; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; C099236:SIX1 protein, human; C104381:EYA1 protein, human; D017027:Protein Tyrosine Phosphatases
keywords: Congenital diaphragmatic hernia (CDH); Eya1; Foetal lung development; Pulmonary hypoplasia; Six1