Eur J Pediatr Surg 2021; 31(01): 120-125
DOI: 10.1055/s-0040-1714736
Original Article

Pbx1, Meis1, and Runx1 Expression Is Decreased in the Diaphragmatic and Pulmonary Mesenchyme of Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia

Toshiaki Takahashi
1  National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
2  Department of Pediatric Surgery, Kansai Medical University, Osaka, Japan
,
Florian Friedmacher
1  National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
3  Department of Pediatric Surgery, University Hospital Frankfurt, Goethe University Frankfurt, Frankfurt am Main, Germany
,
Julia Zimmer
1  National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
4  Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany
,
Prem Puri
1  National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
5  Beacon Hospital, University College Dublin, Dublin, Ireland
› Author Affiliations
Funding This research was financially supported by the National Children's Research Centre and the Children's Medical and Research Foundation.

Abstract

Introduction Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH) are thought to originate from mesenchymal defects in pleuroperitoneal folds (PPFs) and primordial lungs. Pre-B-cell leukemia homeobox 1 (Pbx1), its binding partner myeloid ecotropic integration site 1 (Meis1), and runt-related transcription factor 1 (Runx1) are expressed in diaphragmatic and lung mesenchyme, functioning as transcription cofactors that modulate mesenchymal cell proliferation. Furthermore, Pbx1 −/− mice develop diaphragmatic defects and PH similar to human CDH. We hypothesized that diaphragmatic and pulmonary Pbx1, Meis1, and Runx1 expression is decreased in the nitrofen-induced CDH model.

Materials and Methods Time-mated rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 48) were microdissected on D13, D15, and D18, and were divided into control and nitrofen-exposed specimens. Diaphragmatic and pulmonary gene expression levels of Pbx1, Meis1, and Runx1 were analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence-double-staining for Pbx1, Meis1, and Runx1 was combined with mesenchymal/myogenic markers Gata4 and myogenin to evaluate protein expression.

Results Relative mRNA expression of Pbx1, Meis1, and Runx1 was significantly decreased in PPFs (D13), developing diaphragms/lungs (D15), and muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal-laser-scanning-microscopy revealed markedly diminished Pbx1, Meis1, and Runx1 immunofluorescence in diaphragmatic and pulmonary mesenchyme, associated with less proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared with controls.

Conclusion Decreased Pbx1, Meis1, and Runx1 expression during diaphragmatic development and lung branching morphogenesis may reduce mesenchymal cell proliferation, causing malformed PPFs and disrupted airway branching, thus leading to diaphragmatic defects and PH in the nitrofen-induced CDH model.



Publication History

Received: 20 May 2020

Accepted: 26 June 2020

Publication Date:
30 August 2020 (online)

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