Expression of T-box transcription factors 2, 4 and 5 is decreased in the branching airway mesenchyme of nitrofen-induced hypoplastic lungs.
OBJECTIVE
Pulmonary hypoplasia (PH), characterized by smaller lung size and reduced airway branching, remains a major therapeutic challenge in newborns with congenital diaphragmatic hernia (CDH). T-box transcription factors (Tbx) have been identified as key components of the gene network that regulates fetal lung development. Tbx2, Tbx4 and Tbx5 are expressed throughout the mesenchyme of the developing lung, regulating the process of lung branching morphogenesis. Furthermore, lungs of Tbx2-, Tbx4- and Tbx5-deficient mice are hypoplastic and exhibit decreased lung branching, similar to PH in human CDH. We hypothesized that the expression of Tbx2, Tbx4 and Tbx5 is decreased in the branching airway mesenchyme of hypoplastic rat lungs with nitrofen-induced CDH.


METHODS
Time-mated rats received either nitrofen or vehicle on gestational day 9 (D9). Fetuses were killed on D15, D18 and D21, and dissected lungs were divided into control and nitrofen-exposed specimens. Pulmonary gene expression of Tbx2, Tbx4 and Tbx5 was investigated by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for Tbx2, Tbx4 and Tbx5 was combined with the mesenchymal marker Fgf10 to assess protein expression and localization in branching airway tissue.


RESULTS
Relative mRNA levels of Tbx2, Tbx4 and Tbx5 were significantly reduced in lungs of nitrofen-exposed fetuses on D15, D18 and D21 compared to controls. Confocal laser scanning microscopy showed markedly diminished immunofluorescence of Tbx2, Tbx4 and Tbx5 in mesenchymal cells surrounding branching airways of nitrofen-exposed fetuses on D15, D18 and D21 compared to controls.


CONCLUSIONS
Decreased expression of Tbx2, Tbx4 and Tbx5 in the pulmonary mesenchyme during fetal lung development may lead to a decrease or arrest of airway branching, thus contributing to PH in the nitrofen-induced CDH model.
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, 12, Ireland.
Toshiaki
T
Takahashi
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, 12, Ireland.
Florian
F
Friedmacher
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, 12, Ireland.
Julia
J
Zimmer
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.
Prem
P
Puri
D000015:Abnormalities, Multiple / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D008168:Lung / Q000002:abnormalities* / Q000196:embryology / Q000378:metabolism; D008171:Lung Diseases / Q000235:genetics* / Q000378:metabolism*; D008648:Mesoderm / Q000378:metabolism*; D010647:Phenyl Ethers; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000235:genetics* / Q000378:metabolism*
D010647:Phenyl Ethers; D020825:T-Box Domain Proteins; C103172:T-box transcription factor 5; C007350:nitrofen
Congenital diaphragmatic hernia; Lung branching; Nitrofen; Pulmonary hypoplasia; T-box transcription factor