Pax3 gene expression is not altered during diaphragmatic development in nitrofen-induced congenital diaphragmatic hernia.
OBJECTIVE
Malformations of the pleuroperitoneal folds (PPFs) have been identified as the origin of the diaphragmatic defect in congenital diaphragmatic hernia (CDH). Pax3, expressed in muscle precursor cells (MPCs), plays a key role in regulating myogenesis and muscularization in the fetal diaphragm. Pax3 mutant mice display absence of muscular diaphragm. However, the distribution of muscle precursor cells is reported to be normal in the PPF of the nitrofen-CDH model. We designed this study to investigate the hypothesis that Pax3 gene expression is unaltered in the PPF and developing diaphragm in the nitrofen-induced CDH model.


METHODS
Pregnant rats were treated with nitrofen or vehicle on gestational day (D) 9 and sacrificed on D13, D18, and D21. Pleuroperitoneal folds (D13) and developing diaphragms (D18 and D21) were dissected, total RNA was extracted, and real-time quantitative polymerase chain reaction was performed to determine Pax3 messenger RNA levels. Confocal immunofluorescence microscopy was performed to evaluate protein expression/distribution of Pax3.


RESULTS
Relative messenger RNA expression levels of Pax3 in PPFs and developing diaphragms were not significantly different in the nitrofen group compared with controls. Intensity of Pax3 immunofluorescence was also not altered in PPFs and developing diaphragms of the nitrofen group compared with controls.


CONCLUSIONS
Pax3 gene expression is not altered in the PPFs and developing diaphragm of nitrofen-CDH model, suggesting that the diaphragmatic defect is not caused by disturbance of myogenesis and muscularization.
National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland.
Jan-Hendrik
JH
Gosemann
Takashi
T
Doi
Balazs
B
Kutasy
Florian
F
Friedmacher
Jens
J
Dingemann
Prem
P
Puri
D000818:Animals; D003964:Diaphragm / Q000196:embryology* / Q000378:metabolism / Q000473:pathology; D047108:Embryonic Development / Q000187:drug effects; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D006548:Hernia, Diaphragmatic / Q000139:chemically induced / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D065630:Hernias, Diaphragmatic, Congenital; D008856:Microscopy, Fluorescence; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D010537:Peritoneum / Q000196:embryology / Q000378:metabolism / Q000473:pathology; D010647:Phenyl Ethers / Q000633:toxicity; D010994:Pleura / Q000196:embryology / Q000378:metabolism / Q000473:pathology; D011247:Pregnancy; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction
D000072041:PAX3 Transcription Factor; C530820:PAX3 protein, rat; D051761:Paired Box Transcription Factors; D010647:Phenyl Ethers; D012333:RNA, Messenger; C007350:nitrofen