Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofen-induced congenital diaphragmatic hernia.
OBJECTIVE
Pleuroperitoneal folds (PPFs) are the source of the primordial diaphragm's muscle connective tissue (MCT), and developmental mutations have been shown to result in congenital diaphragmatic hernia (CDH). The protein paired-related homeobox 1 (Prx1) labels migrating PPF cells and stimulates expression of transcription factor 4 (Tcf4), a novel MCT marker that controls morphogenesis of the fetal diaphragm. We hypothesized that diaphragmatic Prx1 and Tcf4 expression is decreased in the nitrofen-induced CDH model.


METHODS
Time-mated rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms were microdissected on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Gene expression levels of Prx1 and Tcf4 were analyzed by qRT-PCR. Immunofluorescence double staining for Prx1 and Tcf4 was performed to evaluate protein expression and localization.


RESULTS
Relative mRNA expression of Prx1 and Tcf4 was significantly downregulated in PPFs (D13), developing diaphragms (D15) and fully muscularized diaphragms (D18) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy revealed markedly diminished Prx1 and Tcf4 expression in diaphragmatic MCT of nitrofen-exposed fetuses on D13, D15, and D18 compared to controls.


CONCLUSIONS
Decreased expression of Prx1 and Tcf4 in the fetal diaphragm may cause defects in the PPF-derived MCT, leading to development of CDH in the nitrofen model.


METHODS
Level 2c (Centre for Evidence-Based Medicine, Oxford).
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.
Toshiaki
T
Takahashi
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.
Julia
J
Zimmer
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.
Florian
F
Friedmacher
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland; Conway Institute of Biomolecular and Biomedical Research, School of Medicine & Medical Science, University College Dublin, Dublin, Ireland. Electronic address: prem.puri@ncrc.ie.
Prem
P
Puri
D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000096:biosynthesis*; D003238:Connective Tissue / Q000002:abnormalities / Q000196:embryology / Q000378:metabolism*; D003964:Diaphragm / Q000002:abnormalities / Q000196:embryology / Q000378:metabolism*; D004195:Disease Models, Animal; D015536:Down-Regulation; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D065630:Hernias, Diaphragmatic, Congenital / Q000139:chemically induced / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000096:biosynthesis*; D010647:Phenyl Ethers / Q000009:adverse effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013723:Teratogens; D000073940:Transcription Factor 4; D014157:Transcription Factors / Q000096:biosynthesis*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D018398:Homeodomain Proteins; C082157:PRRX1 protein, human; D010647:Phenyl Ethers; C532096:TCF4 protein, human; D013723:Teratogens; D000073940:Transcription Factor 4; D014157:Transcription Factors; C007350:nitrofen
Congenital diaphragmatic hernia; Diaphragm; Muscle connective tissue; Nitrofen; Paired-related homeobox 1; Transcription factor 4